Disease severity index for assessment of chronic liver disease

ABSTRACT

A Disease Severity Index (DSI) is provided for assessment of chronic liver disease in a patient using non-invasive liver function test results. A DSI was derived from non-invasive liver function test results based on hepatic blood flow. The DSI is used in methods for prediction of clinical outcomes, prediction of response to antiviral treatment, and assessment of progression of chronic liver diseases. Non-invasive methods to diagnose three distinct categories of patients with Primary Sclerosing Cholangitis (PSC) are provided. The methods can be used to diagnose PSC patients as Slow Progressors, Moderate Progressors and Rapid Progressors.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.14/729,987, filed Jun. 3, 2015; now U.S. Pat. No.: 9,759,731, issuedSep. 12, 2017; which is a Continuation of U.S. patent application Ser.No. 14/078,058, filed Nov. 12, 2013, now U.S. Pat. No.: 9,091,701,issued Jul. 28, 2015; which claims the benefit of U.S. ProvisionalApplication No. 61/725,292, filed Nov. 12, 2012, the entire contents ofeach of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.DK092327 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

A Disease Severity Index (DSI) useful in assessment of chronic liverdisease in a patient is derived from one or more liver function testresults based on hepatic blood flow. The DSI is used in methods forpatient assessment in a number of chronic liver diseases. Non-invasivemethods to diagnose three distinct categories of patients with PrimarySclerosing Cholangitis (PSC) are also provided. The methods can be usedto diagnose PSC patients as Slow Progressors, Moderate Progressors andRapid Progressors.

Description of the Related Art

Until now, fibrosis stage on liver biopsy was considered the goldstandard as the surrogate for clinical outcomes in patients with chronicliver disease. Several studies have demonstrated that severity offibrosis, but not steatosis, predicts future risk for clinical outcome.Unfortunately the accuracy of biopsy in staging of fibrosis iscompromised by both sampling error and variation in histopathologicinterpretation. In addition, biopsy is invasive, costly, not embraced bypatients, and associated with significant risks, including risk oflife-threatening complication or even death. Alternatives to liverbiopsy are desirable.

Chronic Liver Disease. Estimates suggest that 30 million or moreAmericans may be affected by chronic liver disease. Chronic liverdiseases (CLDs) include chronic hepatitis C (CHC), chronic hepatitis B,alcoholic liver disease, Alcoholic SteatoHepatitis (ASH), andNon-Alcoholic Fatty Liver Disease (NAFLD) which can progress from simplefatty liver called steatosis, which is relatively benign, to the moreserious Non-Alcoholic SteatoHepatitis (NASH), autoimmune liver disease,cryptogenic cirrhosis, hemochromatosis, Wilson's disease,alpha-1-antitrypsin deficiency, primary sclerosing cholangitis (PSC) andother cholestatic liver diseases.

All liver diseases have common pathophysiologic characteristics withdisease progression fueled by inflammation, accumulation of fibrosis,and alteration of the portal circulation. Tests involving non-invasiveassessment of portal blood flow are desirable for patients having, orsuspected of having, any chronic liver disease.

Primary Sclerosing Cholangitis. Primary sclerosing cholangitis (PSC) isa progressive liver disease that leads to liver damage and ultimately toliver failure. PSC exhibits inexorable progression but the rate ofprogression varies between patients. Chronic inflammation leads tohardening and scarring of the bile ducts. Liver transplant is the onlyknown cure for PSC, but transplant is typically reserved for patientswith severe liver damage. Patient care involves reducing signs andsymptoms of complications of PSC. The hallmark of PSC pathophysiology isportal fibrosis leading to portal hypertension (PHTN) earlier in diseasecompared to other etiologies of liver disease.

Assessment of disease severity in PSC lacks a gold standard, as liverbiopsy has significant sampling error and is no longer recommended.Hepatic Venous Pressure Gradient (HVPG) is invasive, expensive andimpractical, and clinical models were really created to assesslate-stage disease. Previously disclosed liver function tests SHUNT,Portal HFR and STAT were performed in PSC patients as disclosed inEverson et al., U.S. Ser. No. 13/484,083, filed May 30, 2012, which isincorporated herein by reference. Although these tests could delineatedisease severity, there is still an unmet need for accurate non-invasivemethods for diagnosing rate of progression of PSC.

Two known liver function tests, the Portal HFR (Portal hepaticfiltration rate, FLOW) test and the SHUNT test, have been used toaccurately measure portal blood flow and were previously validated usinga large cohort of patients with chronic hepatitis C. The portal HFR andSHUNT tests for liver function in patients with chronic hepatitis C weredisclosed in prior applications by the present inventors.

The portal HFR (FLOW) test, accurately measures the portal blood flowfrom a minimum of 5 blood samples taken over a period of 90 minutesafter an oral dose of deuterated-cholate. The portal HFR (FLOW) test isdisclosed in Everson, US 2010/0055734, Methods for Diagnosis andIntervention of Hepatic Disorders, filed Sep. 11, 2009, which isincorporated herein by reference.

The SHUNT test, comprises simultaneous administration of an intravenousdose of ¹³C-cholate and an oral dose of deuterated-cholate. The SHUNTtest can be used to measure portal blood flow, and systemic hepaticblood flow and therefore determine the amount of portal-systemicshunting. The SHUNT test is disclosed in Everson et al., US2008/0279766,Methods for Diagnosis and Intervention of Hepatic Disorders, filed Jan.26, 2006, which is incorporated herein by reference. A test forestimating portal blood flow is also applicable to other chronic liverdiseases.

A third test called the STAT test is a screening method for estimatingportal blood flow and hepatic function. The STAT test is disclosed inEverson et al., U.S. Ser. No. 13/484,083, filed May 30, 2012, which isincorporated herein by reference. The STAT test is intended forscreening purposes and is used in conjunction with FLOW and SHUNT teststo monitor hepatic blood flow and hepatic function. For example, apatient with a STAT screening test result above a cut-off level issubjected to the more comprehensive portal HFR and SHUNT tests tomonitor hepatic blood flow and hepatic function in the patient.

The portal HFR (FLOW), SHUNT and STAT tests are currently used fordefining disease severity in patients with chronic hepatitis C and otherchronic liver diseases. A variety of cut-offs have been established foruse in tracking disease progression of specific diseases and assessmentof response to treatments. However, no general index of severity withutility for any chronic liver disease has yet been developed.

The portal HFR and SHUNT tests are valuable tools for assessment ofliver function for a number of clinical applications, for example,selection of patients with chronic hepatitis B who should receiveantiviral therapy; selection of patients with chronic hepatitis C whoshould receive antiviral therapy; assessing the risk of hepaticdecompensation in patients with hepatocellular carcinoma (HCC) beingevaluated for hepatic resection; identifying a subgroup of patients onwaiting list with low MELD (Model for End-stage Liver Disease score) whoare at-risk for dying while waiting for an organ donor; as an endpointin clinical trials; replacing liver biopsy in pediatric populations;tracking of allograft function; measuring return of function in livingdonors; and measuring functional impairment in cholestatic liver disease(PSC, Primary Sclerosing Cholangitis). Although various cut-offs for theFLOW and SHUNT tests have been developed for specific conditions,development of a Disease Severity Index (DSI) applicable to severalclinical conditions in liver disease is clearly desirable.

SUMMARY OF THE INVENTION

In some embodiments, a Disease Severity Index (DSI) is provided for usein methods for monitoring chronic liver disease in a patient. The DSI isderived from liver function test results based on hepatic blood flow.The DSI is used in methods for monitoring treatment and assessment ofdisease severity in a number of chronic liver diseases.

In some embodiments, a method is provided for determining a diseaseseverity index (DSI) value in a patient, the method comprising (a)obtaining one or more liver function test values in a patient having orat risk of a chronic liver disease, wherein the one or more liverfunction test values are obtained from one or more liver function testsselected from the group consisting of SHUNT, portal hepatic filtrationrate (portal HFR), and systemic hepatic filtration rate (systemic HFR);and (b) employing a disease severity index equation (DSI equation) toobtain a DSI value in the patient, wherein the DSI equation comprisesone or more terms and a constant to obtain the DSI value, wherein atleast one term of the DSI equation independently represents a liverfunction test value in the patient, or a mathematically transformedliver function test value in the patient from step; and the at least oneterm of the DSI equation is multiplied by a coefficient specific to theliver function test.

In some embodiments, the method for determining a disease severity index(DSI) value in a patient further comprises comparing the DSI value inthe patient to one or more DSI cut-off values, one or more normalhealthy controls, or one or more DSI values within the patient overtime. In some embodiments, the comparing the DSI value in the patient toone or more DSI cut-off values is indicative of at least one clinicaloutcome. In some embodiments, the clinical outcome is selected from thegroup consisting of Child-Turcotte-Pugh (CTP) increase, varices,encephalopathy, ascites, and liver related death.

In some embodiments, comparing the DSI value within the patient overtime is used to monitor the effectiveness of a treatment of chronicliver disease in the patient, wherein a decrease in the DSI value in thepatient over time is indicative of treatment effectiveness.

In some embodiments, comparing the DSI value in the patient over time isused to monitor the need for treatment of chronic liver disease in thepatient, wherein an increase in the DSI value in the patient over timeis indicative of a need for treatment in the patient.

In some embodiments, the DSI value in the patient is used to monitor theneed for, or the effectiveness of, a treatment of chronic liver diseasein the patient wherein the treatment is selected from the groupconsisting of antiviral treatment, antifibrotic treatment, antibiotics,immunosuppressive treatments, anti-cancer treatments, ursodeoxycholicacid, insulin sensitizing agents, interventional treatment, livertransplant, lifestyle changes, and dietary restrictions, low glycemicindex diet, antioxidants, vitamin supplements, transjugular intrahepaticportosystemic shunt (TIPS), catheter-directed thrombolysis, balloondilation and stent placement, balloon-dilation and drainage, weightloss, exercise, and avoidance of alcohol.

In some embodiments, comparing the DSI value within the patient overtime is used to monitor status or disease progression of a chronic liverdisease in the patient, wherein change in DSI value within the patientover time is used to inform the patient of status of the disease andrisk for future clinical outcomes, wherein an increase in the DSI valuewithin the patient over time is indicative of a worse prognosis, and adecrease in the DSI value within the patient over time is indicative ofa better prognosis.

In some embodiments, at least one term of the DSI equation independentlyrepresents a mathematically transformed liver function test value in thepatient from step wherein the mathematically transformed liver functiontest value in the patient is selected from a log, antilog, natural log,natural antilog, or inverse of the liver function test value in thepatient.

In some embodiments, each term of the DSI equation independentlyrepresents a liver function test value in the patient, or amathematically transformed liver function test value in the patient, andthe at least one term of the DSI equation is multiplied by a coefficientspecific to the liver function test.

In some embodiments, the disease severity index equation isDSI=5.34 (SHUNT)−6.65 (Log_(e) Portal HFR)−8.57 (Log_(e) SystemicHFR)+44.66where SHUNT is SHUNT test value in the patient (%), portal HFR is portalHFR test value in the patient as mL/min/kg, wherein kg is body weight ofthe patient, and systemic HFR is systemic HFR value in the patient asmL/min/kg, wherein kg is body weight of the patient, wherein the SHUNT,the portal HFR, and the systemic HFR test values in the patient wereobtained on the same day.

In some embodiments, the disease severity index equation isDSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50where SHUNT is SHUNT test value in the patient (%), portal HFR is portalHFR test value in the patient as mL/min/kg, wherein kg is body weight ofthe patient, and systemic HFR is systemic HFR value in the patient asmL/min/kg, wherein kg is body weight of the patient, wherein the SHUNT,the portal HFR, and the systemic HFR test values in the patient wereobtained on the same day.

In some embodiments, the DSI value in the patient is used to assesschronic liver disease in the patient selected from chronic hepatitis C,non-alcoholic fatty liver disease or primary sclerosing cholangitis.

In some embodiments, the disease severity index equation used to assesschronic liver disease in the patient isDSI=9.84 (SHUNT)−12.36 LOGe (portal HFR)+50.5where SHUNT is SHUNT test value in the patient (%) and portal HFR isportal HFR test value in the patient as mL/min/kg, wherein kg is bodyweight of the patient, wherein the SHUNT and the portal HFR test valuesin the patient were obtained on the same day. In some embodiments, thechronic liver disease is chronic hepatitis C.

In some embodiments, a SHUNT test value in the patient is used in theDSI equation, and the SHUNT test value is determined by a methodcomprising receiving a plurality of blood or serum samples collectedfrom the patient having PSC, following oral administration of a dose ofa first distinguishable cholate (dose_(oral)) to the patient andsimultaneous intravenous co-administration of a dose of a seconddistinguishable cholate (dose_(iv)) to the patient, wherein the sampleshave been collected over intervals spanning a period of time afteradministration; quantifying the concentration of the first and thesecond distinguishable cholates in each sample; generating anindividualized oral clearance curve from the concentration of the firstdistinguishable cholate in each sample comprising using a computeralgorithm curve fitting to a model oral distinguishable cholateclearance curve and computing the area under the individualized oralclearance curve (AUCoral); generating an individualized intravenousclearance curve from the concentration of the second distinguishablecholate in each sample by use of a computer algorithm curve fitting to amodel intravenous second distinguishable cholate clearance curve andcomputing the area under the individualized intravenous clearance curve(AUCiv); and calculating the shunt value in the patient using theformula:AUC_(oral)/AUC_(iv)×Dose_(iv)/Dose_(oral)×100%.

In some embodiments, the SHUNT test employs a first distinguishablecholate is a first stable isotope labeled cholic acid and a seconddistinguishable cholate is a second stable isotope labeled cholic acid.In some embodiments, the first and second stable isotope labeled cholicacids are selected from 2,2,4,4-d4 cholate and 24-¹³C-cholate. In someembodiments, the samples have been collected from the patient overintervals of from two to seven time points after administration. In someembodiments, the samples have been collected from the patient at 5, 20,45, 60 and 90 minutes after administration. In some embodiments, thesamples have been collected over intervals spanning a period of timefrom the time of administration to a time selected from about 45 minutesto about 180 minutes after administration. In some embodiments, thesamples have been collected over intervals spanning a period of time ofabout 90 minutes or less after administration.

In some embodiments, a portal HFR value in the patient is used in theDSI equation, and the portal HFR value is determined by a methodcomprising the steps of receiving a plurality of blood or serum samplescollected from a patient having or at risk of a chronic liver disease,following oral administration of a dose of a distinguishable cholate(dose_(oral)) to the patient, wherein the samples have been collectedfrom the patient over intervals spanning a period of time afteradministration; measuring concentration of the distinguishable cholatein each sample; generating an individualized oral clearance curve fromthe concentration of the distinguishable cholate in each samplecomprising using a computer algorithm curve fitting to a modeldistinguishable cholate clearance curve; computing the area under theindividualized oral clearance curve (AUC)(mg/mL/min) and dividing thedose (in mg) by AUC of the orally administered stable isotope labeledcholic acid to obtain the oral cholate clearance in the patient; anddividing the oral cholate clearance by the weight of the patient in kgto obtain the portal HFR value in the patient (mL/min/kg).

In some embodiments, a systemic HFR value in the patient is used in theDSI equation and the systemic HFR value in the patient is determined bya method comprising the steps of receiving a plurality of blood or serumsamples collected from a patient having or at risk of a chronic liverdisease, following intravenous administration of a dose of adistinguishable cholate (dose_(iv)) to the patient, wherein the sampleshave been collected from the patient over intervals spanning a period oftime after administration; measuring concentration of thedistinguishable cholate in each sample; generating an individualizedintravenous clearance curve from the concentration of thedistinguishable cholate in each sample comprising using a computeralgorithm curve fitting to a model distinguishable cholate clearancecurve; computing the area under the individualized intravenous clearancecurve (AUC)(mg/mL/min) and dividing the dose (in mg) by AUC of theintravenously administered stable isotope labeled cholic acid to obtainthe intravenous cholate clearance in the patient; and dividing theintravenous cholate clearance by the weight of the patient in kg toobtain the systemic HFR value in the patient (mL/min/kg).

In some embodiments, a method is provided for calculating a diseaseseverity index (DSI) value for a patient suffering from a chronic liverdisease, the method comprising obtaining serum samples from a patientsuffering from a chronic liver disease, wherein the patient previouslyreceived oral administration of a first stable isotope cholate andsimultaneously intravenous administration of a second stable isotopecholate, and wherein blood samples had been collected from the patientover an interval of less than 180 minutes following administration ofthe cholates; assaying the serum samples to calculate the portal hepaticfiltration rate (portal HFR) as mL/min/kg, wherein kg is body weight ofthe patient, the systemic hepatic filtration rate (systemic HFR) asmL/min/kg wherein kg is body weight of the patient, and SHUNT as %; andcalculating a DSI value for the patient by using an equation selectedfrom:DSI=9.84 (SHUNT)−12.36 Log_(e) (portal HFR)+50.5;DSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50; orDSI=5.34 (SHUNT)−6.65 (Log_(e) Portal HFR)−8.57 (Log_(e) SystemicHFR)+44.66.

In some embodiments, the DSI equation isDSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50, and theDSI value is used for identifying increased risk for portal hypertensionor decompensation in the chronic liver disease patient wherein a DSI≥18indicates increased risk for portal hypertension (PHTN), and a DSI≥36indicates an increased risk for decompensation. In some embodiments, theportal hypertension (PHTN) is defined as splemomegaly or varices, anddecompensation is defined as ascites or variceal hemorrhage. In someembodiments, the chronic liver disease is primary sclerosingcholangitis.

In some embodiments, the DSI equation used for calculating a diseaseseverity index (DSI) value for a patient suffering from a chronic liverdisease isDSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50,

and the DSI value is used for prediction of clinical outcomes in thechronic liver disease patient, wherein a DSI≥25 indicates an increasedrisk of clinical outcome in the patient. In some embodiments, thechronic liver disease is chronic hepatitis C. In some embodiments, theclinical outcome is selected from CTP progression, variceal hemorrhage,ascites, hepatic encephalopathy, or liver-related death.

In some embodiments, the patient is on the waiting list for livertransplant (LT), and the DSI equation isDSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50,wherein the DSI value is used for prioritizing the patient on thewaiting list for LT, wherein the priority of the patient on the waitinglist for LT is increased following an increase in the DSI value overtime in the patient, or following a DSI value in the patient of greaterthan 40.

In some embodiments, the DSI equation isDSI=5.34 (SHUNT)−6.65 (Log_(e) Portal HFR)−8.57 (Log_(e) SystemicHFR)+44.66,and the DSI value is used for prediction of future clinical outcomes ina chronic liver disease patient, wherein a DSI>19 indicates an increasedrisk of clinical outcomes in the patient.

In some embodiments, a DSI equation is provided comprising two or moreterms and a constant to obtain the DSI value, wherein at least one termof the DSI equation independently represents a liver function test valuein the patient, or a mathematically transformed liver function testvalue in the patient from step; wherein the at least one term of the DSIequation is multiplied by a coefficient specific to the liver functiontest, and the DSI equation comprises one or more additional termsrepresenting values from clinical biochemistry laboratory assaysselected from the group consisting of serum albumin, alaninetransaminase, aspartate transaminase, alkaline phosphatase, totalbilirubin, direct bilirubin, gamma glutamyl transpeptidase, 5′Nucleotidase, PT-INR (prothrombin time-international normalized ratio),caffeine elimination, antipyrine clearance, galactose eliminationcapacity, formation of MEGX from lidocaine, methacetin-C13, andmethionine-C13; and/or one or more additional terms representingclinical features selected from varices, ascites, or hepaticencephalopathy.

In some embodiments, a method is provided for diagnosing rate ofprogression of primary sclerosing cholangitis (PSC) in a patient, themethod comprising: determining a SHUNT test value or a Portal HFR testvalue in a patient having PSC; employing the SHUNT test value or PortalHFR test value in an algorithm to provide an algorithm result, whereinthe algorithm comprises a term representing the age of the patient inyears at the time of the determining step; and comparing the algorithmresult to a known cut-off value to diagnose the rate of progression ofPSC in the patient. In some embodiments, a method is provided fordiagnosing rate of progression of primary sclerosing cholangitis (PSC)in a patient, wherein if a SHUNT test value (in %) in the patientdivided by the age of the patient is greater than 1.7; then the rate ofprogression of primary sclerosing cholangitis (PSC) in the patient israpid, and the patient having PSC is diagnosed as a Rapid Progressor.

In some embodiments, a method is provided for diagnosing rate ofprogression of primary sclerosing cholangitis (PSC) in a patient,wherein if a SHUNT test value (in %) in the patient divided by the ageof the patient is less than 1.7; and portal HFR (inmL/min/kg)+[0.35×age]>29; then the rate of progression of primarysclerosing cholangitis (PSC) in the patient is slow, and the patienthaving PSC is diagnosed as a Slow Progressor.

In some embodiments, a method is provided for diagnosing rate ofprogression of primary sclerosing cholangitis (PSC) in a patient,wherein if a SHUNT test value (in %) in the patient divided by the ageof the patient is less than 1.7; and portal HFR (inmL/min/kg)+[0.35×age]<29; then the rate of progression of primarysclerosing cholangitis (PSC) in the patient is moderate, and the patienthaving PSC is diagnosed as a Moderate Progressor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the Portal HFR and SHUNT tests. Healthycontrols (upper panel) generally exhibit low SHUNT, high Portal HFR andhigh Systemic HFR; whereas subjects with liver disease (lower panel)such as PSC exhibit higher SHUNT, lower Portal HFR and lower SystemicHFR.

FIG. 2 shows Portal HFR vs. SHUNT test values can distinguish healthycontrols from PSC patients without varices, PSC patients with varicesand PSC patients with decompensation.

FIG. 3 shows Portal HFR vs. age in PSC patients. PHC patients could besegregated into distinct groups based on their Portal HFR test valuesand age at the time of testing.

FIG. 4 shows PSC patients could be segregated into distinct groups basedon their SHUNT test values vs. age at the time of testing.

FIG. 5 shows pro-inflammatory cytokines IFN-γ, TNF-α, GM-CSF, IL-1β,IL-6 and IL-8 from healthy controls and patients with PSC categorized asSlow, Moderate and Rapid Progressors. Each category of patientsexhibited a unique pattern of cytokines suggesting uniquepathophysiological mechanisms.

FIG. 6 shows SHUNT vs. age determines PSC patients categorized as RapidProgressors. The line starting at 0 demarcates SHUNT/age of 1.7;patients above the line exhibiting SHUNT/age>1.7 are categorized asRapid Progressors. Patients with SHUNT/1.7<1.7 are classified into otherPSC categories.

FIG. 7 shows Portal HFR vs. age for PSC patients that have not beenclassified as Rapid Progressors. The line starting at 0 demarcates aPortal HFR+[0.35+age] that is equal to 29. Patients with PortalHFR+[0.35+age] greater than 29 are categorized as Slow Progressors.Patients with Portal HFR+[0.35+age] less than 29 are categorized asModerate Progressors.

FIG. 8 shows STAT test values vs. Age for PSC patients. Most patientscan be categorized using simple cut-offs, shown as dark lines on theplot of Slow, Moderate and Rapid Progressors. Patients between age 30and 40 have overlapping STAT results and are not able to be categorizedby this method.

FIG. 9 shows correlation of log STAT test results at 60 min. vs. logPortal HFR (FLOW) test results for a group of CHC patients. An equationwas derived that could transform the concentration (uM) at 60 min intoan estimated portal flow (mL/min/kg). The equation is y=0.9702x+0.0206,where x is the LOG Portal HFR (FLOW) and y is LOG STAT.

FIG. 10 shows DSI linearly correlates with Ishak fibrosis score (liverbiopsy, left panel) but is not influenced by steatosis (biopsy fatscore, right panel), as provided in Example 9.

FIG. 11 shows performance of DSI in identifying the patients with futureclinical outcomes as compared to that of Ishak fibrosis score (liverbiopsy), platelet count (CBC), and MELD (Model for End-stage LiverDisease score). At the optimum cutoffs, DSI surprisingly outperformedother standard test methods including liver biopsy and MELD forprediction of future clinical outcomes. Specifically, DSI exhibited thehighest sensitivity, specificity, PPV, and NPV when compared to liverbiopsy, platelet count and MELD

FIG. 12 shows ROC curves for predicting outcomes for SHUNT, portal HFRand DSI. Optimum cutoffs were determined as the point on each curveclosest to the top-left corner.

FIG. 13 shows a plot of cholate test results for non-cirrhotic chronichepatitis C patients (Ishak F2,3,4; n=19, 63, 45) with mild disease,moderate disease and severe disease and test results of cholate basedtests SHUNT (%), systemic HFR (mL/min/kg), portal HFR (mL/min/kg) andDSI. Portal HFR is plotted on the X axis and systemic HFR on the Y axis,SHUNT, the ratio of systemic to portal HFR is represented by thediagonal lines, DSI is displayed in shaded regions. Surprisingly,non-cirrhotic patients with high DSI have greater risk of outcomes asdiscussed in Example 10.

FIG. 14 shows a plot of cholate test results for cirrhotic chronichepatitis C patients (Ishak F5, 6; n=48,49) with mild disease, moderatedisease and severe disease test results of cholate based tests SHUNT(%), systemic HFR (mL/min/kg), portal HFR (mL/min/kg) and DSI. PortalHFR is plotted on the X axis and systemic HFR on the Y axis, SHUNT, theratio of systemic to portal HFR is represented by the diagonal lines,DSI is displayed in shaded regions. Surprisingly, cirrhotic patientswith low DSI have lower risk of outcomes as discussed in Example 10.

FIG. 15 shows a plot of cholate test results for primary sclerosingcholangitis patients and healthy controls. Portal HFR is plotted on theX axis and systemic HFR on the Y axis, SHUNT, the ratio of systemic toportal HFR is represented by the diagonal lines, DSI is displayed inshaded regions. Predictive DSI cutoffs for PSC disease, varices, anddecompensation are shown at the interfaces between zones.

FIG. 16 shows a comparison of DSI values with MELD for PSC patients.Panel A shows a comparison of DSI with MELD for all the PSC patients.The DSI of healthy controls is also shown for reference. Panel B shows aDSI of approximately 20 clearly separates the PSC patients with varicesfrom the PSC patients without varices. Panel C shows a DSI ofapproximately 35 clearly separates the PSC patients with decompensationfrom those without decompensation.

FIG. 17 shows changes in DSI and disease severity in PSC patients thatwere assessed after a 1 year follow-up. The change in DSI was plottedagainst the age of the patient. Patients with DSI values indicative ofslow PSC progression are shown in the left panel where a cutoff=18 isindicative of PHTN. Patients with DSI values indicative of moderate andrapid PSC progression are shown in the right panel where a cutoff=36 isindicative of decompensation.

FIG. 18 shows a plot of DSI versus MELD scores in PSC patients on thewaiting list for liver transplantation. DSI was superior to MELD inassessing risk for complications and priority for liver transplant inPSC patients. Despite low MELD scores, PSC patients with DSI>20developed portal hypertension-related complications, and PSC patientswith DSI>40 required liver transplantation.

FIG. 19A shows a graph of patients achieving SVR compared to quartilesfor hepatic function. The probability of SVR correlated best with DSI.

FIG. 19B shows hepatic functional improvement in SHUNT, portal HFR andDSI, from left to right, after SVR following retreatment of chronic HCVpatients with PEG/RBV (peginterferon/ribavirin). More severe baselineimpairment resulted in greater functional improvement after SVR whentested two years after baseline.

DETAILED DESCRIPTION OF THE INVENTION

The methods and tests disclosed herein are based on a new view ofchronic liver disease, that it is the disruption of the portal bloodflow, not fibrosis per se, that is deleterious and should be targetedfor analysis of liver function.

Previously disclosed liver function tests, the Portal HFR (Portalhepatic filtration rate, FLOW) test and the SHUNT test, are used toaccurately measure portal blood flow and were previously validated usinga large cohort of patients with chronic hepatitis C. The portal HFR andSHUNT tests for liver function in patients with chronic hepatitis C weredisclosed in prior applications by the present inventors.

In some embodiments, it has been surprisingly found that when either theportal HFR result or the SHUNT result was divided by age, the functionalassessment was able to define categories of disease in PSC. PSC patientssegregated into distinct groups based on their Portal HFR and SHUNTvalues and age at the time of testing. Slow PSC patients had only modestdeclines in function compared to controls. Moderate and rapid PSCpatients exhibited more complications and at earlier ages. Slow,Moderate and Rapid Progressors could be differentiated by using eitherportal HFR divided by age (FIG. 3) or SHUNT divided by age (FIG. 4). Tothe best of the inventor's knowledge, this is the first time thatfunctional assessment defined categories of disease in PSC.

Chronic liver diseases (CLDs) are all characterized by a similarpathophysiology with inflammation, cell death, and fibrosis leading to aprogressive disruption of the hepatic microvasculature so a test tomeasure portal blood flow will work for assessment of all CLDs.

Almost all the other proposed tests to assess chronic liver disease havefocused on fibrosis, either on serum biomarkers or the change in tissueelasticity (Mukherjee and Sorrell, 2006, Noninvasive tests for liverfibrosis. Semin Liver Dis. 26: 337-347; Manning and Afdhal, 2008.Diagnosis and quantitation of fibrosis. Gastroenterology. 134:1670-1681; Poynard et al., 2008, Concordance in a world without a goldstandard: A new non-invasive methodology for improving accuracy offibrosis markers. PLoS One. 3: e3857).

Both fibrosis and microvasculature disruption do increase as diseaseprogresses but they are not perfectly linked. This explains why patientswith extreme fibrosis, cirrhosis, can remain stable as long as theirportal flow is maintained above a critical threshold. It also explainswhy those patients with only moderate fibrosis but severely impairedflow can have serious complications. This new insight can change thewhole focus of liver disease assessment. By targeting the portal flowphysicians can easily detect early stage liver disease, accuratelyassess the status of their patients, and predict clinical outcomes. Moreeffective treatments for liver disease can result from having researchon new therapies and new drugs focus on improving and/or maintaining theportal flow.

Chronic Hepatitis C. Hepatitis C is an infectious disease affecting theliver and caused by the hepatitis C virus (HCV). HCV infection can goundetected for many years and is often asymptomatic. However, chronicinfection can lead to scarring of the liver, cirrhosis and liverfailure, liver cancer or life-threatening esophageal and gastricvarices. Patients with cirrhosis or liver cancer may require a livertransplant, although the virus can reoccur after transplantation.Standard therapy includes peginterferon with ribavirin, and clinicaltrials involving further combination with bocepravir or telepravir areongoing. Globally, about 180 million people are infected with HCV.Rosen, Chronic Hepatitis C Infection, N Engl J Med 2011; 364:2429-38.

There are a number of diagnostic tests available to detect HCV infectionincluding HCV antibody enzyme immunoassay (ELISA), recombinantimmunoblot assay, and HCV RNA polymerase chain reaction (PCR). However,chronic infections are typically asymptomatic and are most oftendiscovered following investigation of elevated liver enzyme levels orduring routine screening. Unfortunately, diagnostic testing cannotdistinguish between acute and chronic cases. In addition, liver enzymesare poorly correlated with disease severity. Liver biopsies are used todetermine the degree of liver damage present, but there are risks fromthe procedure. Better non-invasive tests for liver function haverecently been developed.

Two known liver function tests, the Portal HFR (Portal hepaticfiltration rate, FLOW) test and the SHUNT test, have been used toaccurately measure portal blood flow and were previously validated usinga large cohort of patients with chronic hepatitis C. The portal HFR andSHUNT tests for liver function in patients with chronic hepatitis C weredisclosed in prior applications by the present inventors.

Nonalcoholic Fatty Liver Disease. Non-Alcoholic Fatty Liver Disease(NAFLD) (Browning et al., 2004, Prevalence of hepatic steatosis in anurban population in the United States: Impact of ethnicity. Hepatology.40: 1387-1395) may affect up to one-third of the US population and thisvast epidemic is mostly hidden because people are usually asymptomaticand have normal ‘liver function tests’—clinical biochemistry laboratoryblood assays such as serum albumin, alanine transaminase, aspartatetransaminase, alkaline phosphatase, total bilirubin, direct bilirubin,and gamma glutamyl transpeptidase. The prevalence of NAFLD continues torise along with the major risk factors which are obesity, metabolicsyndrome, and insulin resistance. NAFLD can progress from simple fattyliver called steatosis, which is relatively benign, to the more seriousNASH, Non-Alcoholic SteatoHepatitis. Hepatitis is inflammation of theliver and can also be caused by excessive drinking, as in AlcoholicSteatoHepatitis (ASH), or viral infection, i.e., Chronic Hepatitis C(CHC). All these chronic liver diseases (CLDs) are characterized by asimilar patho-physiology with inflammation, cell death, and fibrosisleading to a progressive disruption of the hepatic microvasculature.About 5% of NAFLD patients will progress to cirrhosis (Adams et al.,2005, The natural history of nonalcoholic fatty liver disease: Apopulation-based cohort study.

Gastroenterology. 129: 113-121) and NAFLD will surpass CHC as theleading indication for liver transplantation.

Difficulties in Monitoring Patients with Chronic Liver Disease.Currently the only way to distinguish Non-Alcoholic SteatoHepatitis(NASH) from steatosis and to monitor NASH progression is through aneedle biopsy, which assesses the grade of inflammatory activity and thestage of fibrosis. Biopsy is considered the gold standard despitesuffering from numerous sources of inaccuracy and the risks of aninvasive procedure. Patients must be sedated and a portion willexperience bleeding and other complications (Janes and Lindor, 1993, AnnIntern Med. 118: 96-98; Seeff et al., 2010, Clin Gastroenterol Hepatol.8: 877-883). The needle biopsy is a very small specimen of a very largeorgan and it is very difficult to obtain large enough pieces from enoughlocations for adequate sampling (Vuppalanchiet al., 2009, ClinGastroenterol Hepatol. 7: 481-486; Bedossa et al., 2003, Hepatology. 38:1449-1457; Regev et al., 2002, Am J Gastroenterol. 97: 2614-2618).Biopsy interpretation is subjective and depends on the expertise of theobserver (Rousselet et al., 2005, Hepatology. 41: 257-264) and the sizeand number of tissue samples (Rousselet et al., 2005; Vuppalanchi etal., 2009). In describing the progression of fibrosis in CHC the 6 stageIshak system (Ishak et al., 1995, J Hepatol. 22: 696-699) may be used,but more typical is a simpler 4 stage system (Knodell et al., 1981,Hepatology. 1: 431-435; Batts and Ludwig, 1995, Am J Surg Pathol. 19:1409-1417; Scheuer, 1991, J Hepatol. 13: 372-374) such as Metavir(Group, TFMCS, 1994, Hepatology. 20: 15-20) which is very comparable tothe 4 stage system used for NASH (Brunt et al., 1999, Am JGastroenterol. 94: 2467-2474; Kleiner et al., 2005. Hepatology 41:1313-1321). However, the heterogeneity of lesions in NASH decreases theaccuracy (Ratziu et al., 2005, Gastroenterology. 128: 1898-1906). It isnot practical to biopsy a third of the population especially since themethod has an estimated error rate of 20% or greater. Other standardliver blood tests are not very useful. Liver enzymes such as ALT or ASTmay spike during activity flares, but often they are in the normal rangedue to the slow rate of progression. The liver's production of albuminor clotting factors only declines at the latest stages of CLD.Noninvasive means to distinguish NASH from steatosis and accuratelymonitor NASH progression are desirable.

Deficiencies of Other Non-invasive Test Methods. The need fornon-invasive liver assessment has led to the commercialization of newmethods by others including biomarker panels, metabolic breath tests,and transient elastography. Each of these other non-invasive testmethods suffers from disadvantages.

Biomarker panels (Mukherjee and Sorrell, 2006, Noninvasive tests forliver fibrosis. Semin Liver Dis. 26: 337-347; Shah et al., 2009,Comparison of noninvasive markers of fibrosis in patients withnonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 7:1104-1112) such as FibroTest® are not sensitive enough to detect eitherearly stage CHC (Boursier et al., 2009. Improved diagnostic accuracy ofblood tests for severe fibrosis and cirrhosis in chronic hepatitis c.Eur J Gastroenterol Hepatol. 21: 28-38; Shaheen et al., 2007, Fibrotestand fibroscan for the prediction of hepatitis c-related fibrosis: Asystematic review of diagnostic test accuracy. Am J Gastroenterol. 102:2589-2600) or NASH (Ratziu et al., 2006, Diagnostic value of biochemicalmarkers (fibrotest-fibrosure) for the prediction of liver fibrosis inpatients with non-alcoholic fatty liver disease. BMC Gastroenterol. 6:6; Angulo et al., 2007, The NAFLD fibrosis score: A noninvasive systemthat identifies liver fibrosis in patients with NAFLD. Hepatology. 45:846-854; Wong et al., 2010, Diagnosis of fibrosis and cirrhosis usingliver stiffness measurement in nonalcoholic fatty liver disease.Hepatology. 51: 454-462) or to track progression because circulatingproteins/fragments can't report accurately on fine structure, thedisruption of the microvasculature, and impairment of flow.

Metabolic breath tests are variable because they rely on cytochrome P450(CYP) enzymes which vary according to gender, age, genetics, diet,medications and they are insensitive to early stage disease because theenzymes do not significantly decline until later stages. BreathID® has amethacetin metabolic test in FDA trials, but this method failed todetect early stage CHC in earlier studies (Braden et al., 2005.¹³C-methacetin breath test as liver function test in patients withchronic hepatitis c virus infection. Aliment Pharmacol Ther. 21:179-185).

FibroScan®, also in FDA trials, uses transient elastography to measureliver stiffness to estimate fibrosis (Del Poggio and Colombo, 2009. Istransient elastography a useful tool for screening liver disease? WorldJ Gastroenterol. 15: 1409-1414). This method is insensitive to earlystage CLD (Del Poggio and Colombo, 2009, infra; Friedrich-Rust et al.,2008. Performance of transient elastography for the staging of liverfibrosis: A meta-analysis. Gastroenterology. 134: 960-974) including CHC(Shaheen et al., 2007, infra, and Rossi et al., 2003. Validation of thefibrotest biochemical markers score in assessing liver fibrosis inhepatitis c patients. Clin Chem. 49: 450-454) or NASH (Wong et al.,2010, Diagnosis of fibrosis and cirrhosis using liver stiffnessmeasurement in nonalcoholic fatty liver disease. Hepatology. 51:454-462) and is compromised by obesity, a major risk factor for NAFLD.More effective noninvasive means to distinguish NASH from steatosis andaccurately monitor NASH progression are clearly needed.

The new focus on portal flow could revolutionize how chronic liverdisease is staged and monitored. Biopsy would still be useful in theinitial diagnosis to rule out auto-immune disease and inheriteddisorders but would not be used to assess patients' status or followthem over time. Impairment of portal flow would be used to guidemanagement and determine when it would be appropriate to screen forvarices and hepatocellular carcinoma. Portal flow would be a new moreaccurately determined endpoint for clinical trials.

A schematic of the previously disclosed portal HFR and SHUNT tests isshown in FIG. 1. The oral cholate clearance (dose/area under oralclearance curve) is a measure of the effective portal blood flow. Theoral clearance per kg body weight is used to determine the portal HFR.The IV cholate clearance (dose/area under IV clearance curve) is ameasure of the total hepatic blood flow. The IV clearance per kg bodyweight determines the systemic HFR. The ratio of IV to oral clearancesassesses the portal-systemic shunt fraction (SHUNT). In one aspect, thedisclosure provides methods wherein the oral cholate clearance or portalHFR can be estimated from the oral cholate serum concentration at asingle time point, for example, at 60 minutes after administration(STAT).

A schematic of the portal HFR and SHUNT tests is shown in FIG. 1. FIG. 1shows that healthy controls (upper panel) generally exhibit low SHUNT,high portal HFR and high Systemic HFR; whereas subjects with liverdisease (lower panel) such as PSC exhibit higher SHUNT, lower portal HFRand lower systemic HFR.

In health, the orally administered deuterated cholate is delivered tothe liver via the portal circulation. Its clearance is a measure of theportal circulation—hence the designation Portal HFR. The intravenouslyadministered ¹³C-cholate is delivered to the liver via both hepaticarterial and portal venous circulations—hence the designation SystemicHFR. SHUNT is a ratio of Systemic HFR to Portal HFR. The normal rangesfor these tests are shown in the top panels.

With disease—SHUNT increases and both portal and systemic HFRdecrease—as shown in the bottom panels.

For example, normal healthy controls typically exhibit SHUNT (IV cholateclearance/oral cholate clearance) of about 20%, portal HFR (oral cholateclearance per kg body weight) of about 30 mL/min/kg, and systemic HFR(intravenous cholate clearance per kg body weight) of about 6 mL/min/kg.Liver disease patients typically exhibit higher SHUNT values of betweenfrom about 30% to 90%. Liver disease patients typically exhibit lowerportal HFR of from about 20 mL/min/kg to about 2 mL/min/kg. Liverdisease patients typically exhibit lower systemic HFR of from about 4mL/min/kg to about 1 mL/min/kg.

In the diseased liver, as more blood escapes extraction by intra- andextra-hepatic shunting to the systemic circulation, the SHUNT increases,HFR or portal flow decreases, and STAT increases. In a normal controlsubject, the effective portal blood flow (portal HFR, FLOW) is high in ahealthy liver due to low vascular resistance. Portal-systemic shunting(SHUNT) is minimal. Oral cholate at 60 min (STAT) is low. For example,in a healthy control FLOW=37 mL min⁻¹ kg⁻¹, SHUNT=18% and STAT=0.2 μM.However, in a subject with liver disease, inflammation, fibrosis, andincreased vascular resistance reduce the effective portal blood flow(FLOW). Portal-systemic shunting (SHUNT) is increased. Oral cholate at60 min (STAT) is high. For example in a CHC F2 patient, FLOW=9 mL min⁻¹kg⁻¹, SHUNT=35% and STAT=1.6 μM.

Portal HFR (FLOW) and SHUNT tests are used to determine portal bloodflow and liver function, for example, in healthy controls and patientswith chronic hepatitis C; these tests are disclosed in US 2010/0055734and US2008/0279766, which are each incorporated herein by reference. TheSTAT test was developed as a screening test and is utilized to estimateportal blood flow and screen large populations for detection of patientswith chronic liver disease, including chronic hepatitis C, PSC andNAFLD. The STAT test was developed to estimate portal blood flow andscreen large populations for detection of patients with chronic liverdisease, including chronic hepatitis C, PSC and NAFLD. The relationshipof STAT to prior art methods of determining clearance of cholate fromthe portal circulation, specifically the FLOW and SHUNT tests, has beenvalidated using a large cohort of patients with chronic hepatitis C. TheSTAT test is disclosed in U.S. Ser. No. 13/484,083, filed May 30, 2012,which is incorporated herein by reference.

In some embodiments, the portal HFR value in the patient is estimatedfrom a STAT test value in the subject, wherein the STAT test value inthe subject is obtained by a method comprising (a) receiving a singleblood or serum sample collected from the subject having PSC, followingoral administration of a dose of a distinguishable cholate compound(dose_(oral)) to the subject, wherein the sample has been collected fromthe subject at a specific time point within about 20-180 minutes afteradministration; (b) measuring concentration of the distinguishablecholate compound in the sample.

In some embodiments, the systemic HFR value in the patient is determinedby a method comprising (a) receiving a plurality of blood or serumsamples collected from a patient having or at risk of a chronic liverdisease, following intravenous administration of a dose of adistinguishable cholate (dose_(oral)) to the patient, wherein thesamples have been collected from the patient over intervals spanning aperiod of time after administration; (b) measuring concentration of thedistinguishable cholate in each sample; (c) generating an individualizedintravenous clearance curve from the concentration of thedistinguishable cholate in each sample comprising using a computeralgorithm curve fitting to a model distinguishable cholate clearancecurve; (d) computing the area under the individualized oral clearancecurve (AUC)(mg/mL/min) and dividing the dose (in mg) by AUC of theintravenously administered stable isotope labeled cholic acid to obtainthe intravenous cholate clearance in the patient; and (e) dividing theintravenous cholate clearance by the weight of the patient in kg toobtain the portal HFR value in the patient (mL/min/kg).

In some embodiments, the single blood or serum sample in the STAT testis collected at one single time point selected from about 20. 25, 30,35, 40, 45, 50, 55, 50, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180minutes, or any time point in between, after oral administration of thedistinguishable cholate compound.

In some embodiments, the single blood or serum sample in the STAT testis collected at one time point selected from about 45, about 60 or about90 minutes after oral administration of the distinguishable cholatecompound.

In some embodiments, the single blood or serum sample is collected atabout 60 minutes after oral administration of the distinguishablecholate compound.

In some embodiments, the single blood or serum sample is collected atabout 45 minutes after oral administration of the distinguishablecholate compound.

In some embodiments, the single blood or serum sample is collected atabout 90 minutes after oral administration of the distinguishablecholate compound.

In some embodiments, the estimated hepatic blood flow (HBF) iscalculated with the following equation:HBF=(Cholate clearance after intravenousadministration)/[1−(SHUNT/100))×(1−(Hematocrit %/100))]

Previously, human studies demonstrated the clinical utility of FLOW andSHUNT testing in CHC. A number of new liver tests have been proposedover the years but there have been few studies to directly compare theirefficacy and actual clinical utility. A very large multicenter HALT-Ctrial was conducted whose main objective was to determine the efficacyof long term hepatitis C virus suppression but which also included anancillary study to evaluate a battery of new quantitative liver functiontests. (Everson et al., 2009. Quantitative tests of liver functionmeasure hepatic improvement after sustained virological response:Results from the HALT-C trial. Aliment Pharmacol Ther. 29: 589-601).Nearly 300 patients with advanced (Ishak F2-6) but compensated CLD weretested. A recently completed Early CHC study compared these tests in 25healthy controls and 23 early stage (Ishak F1-2) CHC patients in orderto examine the entire spectrum of this CLD. The liver's metaboliccapacity was assessed using caffeine, antipyrine, lidocaine, andgalactose tests. All these activities were reduced in patients withcirrhosis, but none were different in early stage CHC patients comparedto healthy controls. (Everson et al., 2008. The spectrum of hepaticfunctional impairment in compensated chronic hepatitis c: Results fromthe hepatitis c anti-viral long-term treatment against cirrhosis trial.Aliment Pharmacol Ther. 27: 798-809). These results suggest thatmetabolic capacity is maintained until there is significant loss offunctional parenchyma in later stage CLD. In HALT-C the patients weretested serially every 2 years and followed to monitor outcomes. FLOW,using a cutoff of <9.5 ml/min/kg, was superior to the other tests inpredicting clinical outcomes with the highest sensitivity, specificity,positive predictive value (PPV), negative predictive value (NPV) and thebest performance by ROC analysis (Quantitative Liver Function TestsImprove the Prediction of Clinical Outcomes in Chronic Hepatitis C:Results from the HALT-C Trial, Everson et al, submitted toGastroenterology). FLOW had a higher ROC c statistic (0.84) relative toSHUNT (0.79). The improvement after SVR was more significant for FLOW(p=0.0002) than for SHUNT (p=0.0003) (Everson et al., 2009, infra). Inthe Early CHC study, FLOW decreased from 34±14 ml/min/kg (mean±SD) incontrols to 23±10 ml/min/kg in early CHC (p<0.002) but the increase inSHUNT (20±6% in controls vs, 31±14% in early CHC patients p<0.0002) wasmore statistically significant. None of the other tests coulddistinguish early stage CHC patients from healthy controls. Theseresults suggest that SHUNT and FLOW outperform other functional tests indetecting early liver disease, tracking patients, and predictingclinical outcomes.

Disease Severity Index (DSI)

Although various direct cut-offs for the FLOW and SHUNT tests werepreviously developed for specific conditions, in some cases use of aDisease Severity Index (DSI) more clearly delineates patient categoriesin chronic liver disease.

The “Disease Severity Index” (DSI) employs a mathematical model designedfor adaptation of a bioassay result (liver function test) to theassessment of disease severity of an individual patient. For example, aDSI equation is developed using liver function test results from adefined patient population and healthy controls. In some embodiments, aDSI equation is developed from a specific patient population. The DSIequation has one or more terms selected from SHUNT, Portal HFR, and/orSystemic HFR depending on type or severity of liver disease. In someembodiments, one or more DSI cut-offs are used for DSI comparison,depending on type of disease and severity of disease. In someembodiments, use of the DSI in a patient requires only a simple tablelook up.

In some embodiments, a method of determining disease severity index(DSI) in a patient with chronic liver disease comprises (a) obtainingone or more liver function test values in a patient having a chronicliver disease, wherein the one or more liver function test values areobtained from one or more liver function tests selected from the groupconsisting of SHUNT, Portal HFR and Systemic HFR; and (b) employing adisease severity index equation (DSI equation) comprising one or moreterms and a constant to obtain the DSI; where at least one term of theDSI equation independently represents a liver function test value in thepatient, or a mathematically transformed liver function test value inthe patient; and the at least one term of the DSI equation is multipliedby a coefficient specific to the liver function test. In someembodiments, the mathematically transformed liver function test value inthe patient is selected from a log, antilog, natural log, naturalantilog, or inverse of the liver function test value in the patient. Insome embodiments, each term of the DSI equation represents a liverfunction test value or a mathematically transformed liver function testvalue.

The constant and coefficient(s) of the DSI equation can vary with liverdisease type and/or disease severity. In some embodiments, the constantand coefficients are interrelated so, for example, if all were dividedby 10 then the DSI would go from 0-5, rather than 0-50, and healthywould be 1 instead of 10. In some embodiments, the constant is apositive number between 5 and 125. In some embodiments, the SHUNTcoefficient is a number between 0 and positive 25. In some embodiments,the Portal HFR coefficient is a number between 0 and negative 25. Insome embodiments, the Systemic HFR coefficient is a number between 0 andnegative 25.

In some embodiments, the at least one term in the DSI equation ismultiplied by a coefficient specific to each type of test, to obtain theDSI. In some embodiments, the DSI in the patient is compared to one ormore DSI cut-off values indicative of at least one clinical outcome.

In some embodiments, the disclosure provides a method of determiningdisease severity index (DSI) in a patient with chronic liver disease,the method comprising (a) obtaining a SHUNT test value and a Portal HFRtest value from a patient having a chronic liver disease; and (b)employing a disease severity equation comprising a first term for theSHUNT test value and a second term for the Portal HFR value to obtainthe DSI.

In a specific embodiment, a DSI equation was developed by use of cholatetesting that was performed at baseline in 224 chronic HCV patients(Ishak F2-F6) enrolled in the HALT-C clinical trial, characterized byCTP scores of 5 or 6 and no prior history of clinical complications.

Specifically, archive serum was re-analyzed to determine cholateclearance curves for systemic Hepatic Filtration Rate (HFR) fromclearance of intravenously administered cholate, Portal HFR from orallyadministered cholate, and SHUNT from the ratio of clearances using animproved LCMS method validated to FDA guidelines. Patients were followedfor clinical outcomes for up to 8.3 years (4.9±2.2 years, mean±SD).Clinical outcomes (n=54) were defined as CTP progression, varicealhemorrhage, ascites, hepatic encephalopathy, or liver-related death.

Derivation of a Disease Severity Index (DSI) was performed usingunivariate Cox univariate Cox proportional hazard regression analysis asshown in Example 10, Table 13. Ageneric equation was developed usingSHUNT, portal HFR and systemic HFR from cholate testing.DSI=A(SHUNT)+B(log_(e) portal HFR)+C(log_(e) systemic HFR)+D.

A DSI equation was developed based on prediction of first clinicaloutcome in the HALT-C cohort:DSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50.

Surprisingly, although this DSI equation was developed in a cohort ofHCV patients, it has provided accurate assessment of disease severity inother chronic liver diseases primary sclerosing cholangitis (PSC) andnon-alcoholic fatty liver disease (NAFLD), for example, as provided in,for example, Examples 9-13.

In some embodiments, a cholate test based DSI is used in a method todifferentiate PSC patients from healthy controls, listed PSC patientsfrom PSC patients not listed for liver transplant, and listed patientswith varices from those without varices, as shown in Example 12.

In some embodiments, a DSI value in a PSC patient based on dual oral andintravenous cholate clearances may be superior to MELD score inassessing the risks for complications and priority for liver transplantin PSC.

In some embodiments, cholate testing and DSI is used in a method foridentifying chronic liver disease patients at risk for portalhypertension and/or decompensation, where portal hypertension (PHTN) isdefined as splemomegaly or varices, and decompensation is defined asascites or variceal hemorrhage.

In some embodiments, the chronic liver disease is selected from chronichepatitis C (CHC), chronic hepatitis B, alcoholic liver disease,Alcoholic SteatoHepatitis (ASH), and Non-Alcoholic Fatty Liver Disease(NAFLD) which can progress from simple fatty liver called steatosis,which is relatively benign, to the more serious Non-AlcoholicSteatoHepatitis (NASH), autoimmune liver disease, cryptogenic cirrhosis,hemochromatosis, Wilson's disease, alpha-1-antitrypsin deficiency,primary sclerosing cholangitis (PSC) and other cholestatic liverdiseases.

In some embodiments, a method for determining a disease severity index(DSI) value in a patient is provided, the method comprising obtainingserum samples from a patient suffering from a chronic liver disease,wherein the patient previously received oral administration of a firststable isotope cholate and simultaneously intravenous administration ofa second stable isotope cholate, and wherein blood samples had beencollected from the patient over an interval of less than 180 minutesfollowing administration of the cholates; assaying the serum samples tocalculate the portal hepatic filtration rate (portal HFR) as mL/min/kg,wherein kg is body weight of the patient, the systemic hepaticfiltration rate (systemic HFR) as mL/min/kg wherein kg is body weight ofthe patient, and SHUNT as %; and calculating a DSI value for the patientby using the equation:DSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50.

In some embodiments, the DSI value is used in a method for identifyingincreased risk for portal hypertension or decompensation in the chronicliver disease patient wherein a DSI greater than 18 indicates increasedrisk for portal hypertension (PHTN), and a DSI greater than 36 indicatesan increased risk for decompensation, where portal hypertension (PHTN)is defined as splemomegaly or varices, and decompensation is defined asascites or variceal hemorrhage.

In some embodiments, the DSI value is used in a method for prediction ofclinical outcomes in the chronic liver disease patient, wherein a DSI>25indicates an increased risk of clinical outcome in the patient. In someembodiments, the chronic liver disease is chronic hepatitis C and theclinical outcome is selected from CTP progression, variceal hemorrhage,ascites, hepatic encephalopathy, or liver-related death.

In some embodiments, the DSI value is used in a method for prediction ofsustained virological response in a patient suffering from chronichepatis B or chronic hepatitis C following antiviral treatment, whereindecrease in DSI value in the patient over time following antiviraltreatment is indicative of sustained virological response.

In some embodiments, the DSI value in a patient having a chronic liverdisease is used for prioritizing the patient on the waiting list forliver transplant (LT), comprising increasing the priority of the patienton the waiting list for LT following an increase in the DSI value overtime in the patient, or following a DSI value of greater than 40 whenthe DSI value is obtained according to the equation:DSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50where SHUNT is SHUNT test value in the patient (%), portal HFR is portalHFR test value in the patient as mL/min/kg, wherein kg is body weight ofthe patient, and systemic HFR is systemic HFR value in the patient asmL/min/kg, wherein kg is body weight of the patient, wherein the SHUNTand the portal HFR test values in the patient were obtained on the sameday.

In some embodiments, the DSI value in a patient having a chronic liverdisease is used in a method for prediction of future clinical outcome oridentifying patients with medium/large varices, when the DSI value inthe patient is obtained according to the equation:DSI=5.34 SHUNT−6.65 Log_(e) Portal HFR−8.57 Log_(e) Systemic HFR+44.66wherein a DSI>19 indicates high risk of medium to large varices; DSI10-19 is indicative of low risk of medium/large varices; and DSI of 0-10is indicative of healthy liver function; and a DSI>19 indicates highrisk of clinical outcomes; DSI 10-19 is indicative of low risk ofclinical outcomes; and DSI of 0-10 is indicative of healthy liverfunction.

In some embodiments, the chronic liver disease is selected from chronichepatitis C, non-alcoholic fatty liver disease (NAFLD), or primarysclerosing cholangitis (PSC).

In some embodiments, a method is provided for identifying increased riskfor portal hypertension or decompensation in a chronic liver diseasepatient, the method comprising obtaining serum samples from a patientsuffering from a chronic liver disease, wherein the patient previouslyreceived oral administration of a first stable isotope cholate andsimultaneously intravenous administration of a second stable isotopecholate, and wherein blood samples had been collected from the patientover an interval of less than 180 minutes following administration ofthe cholates; assaying the serum samples to calculate the portal hepaticfiltration rate (portal HFR) as mL/min/kg, wherein kg is body weight ofthe patient, the systemic hepatic filtration rate (systemic HFR) asmL/min/kg wherein kg is body weight of the patient, and SHUNT as %;calculating a DSI value for the patient by using the equation:DSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50,wherein a DSI greater than 18 indicates increased risk for portalhypertension (PHTN), and a DSI greater than 36 indicates an increasedrisk for decompensation.

In some embodiments, a method is provided for calculating a diseaseseverity index (DSI) value for a patient, the method comprisingobtaining serum samples from a patient suffering from or at risk of achronic liver disease, wherein the patient previously received oraladministration of a first stable isotope cholate and simultaneouslyintravenous administration of a second stable isotope cholate, andwherein blood samples had been collected from the patient over aninterval of less than 180 minutes following administration of thecholates; assaying the serum samples to calculate the portal hepaticfiltration rate (portal HFR) as mL/min/kg, wherein kg is body weight ofthe patient, the systemic hepatic filtration rate (systemic HFR) asmL/min/kg wherein kg is body weight of the patient, and SHUNT as %;calculating a DSI value for the patient by using the equation:DSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50.

In some embodiments, the SHUNT, systemic HFR, and portal HFR test valuesin the patient are obtained on the same day.

In some embodiments, cholate testing DSI is used for adjust the priorityfor liver transplantation for chronic liver disease patients on thewaiting list.

In some embodiments, cholate testing DSI is used to adjust the priorityfor liver transplantation for chronic liver disease patients on thewaiting list, wherein the chronic liver disease is chronic hepatitis C,NAFLD or primary sclerosing cholangitis (PSC).

In another specific embodiment, a DSI equation was developed usingportal HFR and SHUNT test values from HCV patients:DSI=9.84 (SHUNT)−12.36 LOGe (portal HFR)+50.5where SHUNT is SHUNT test value in the patient and portal HFR is portalHFR test value in the patient. In some embodiments, the SHUNT and theportal HFR test values in the patient were obtained on the same day.

In some embodiments, a DSI equation has one or more additional termsrepresenting a different clinical biochemistry laboratory blood assayssuch as serum albumin, alanine transaminase, aspartate transaminase,alkaline phosphatase, total bilirubin, direct bilirubin, gamma glutamyltranspeptidase, 5′ Nucleotidase, PT-INR (prothrombin time-internationalnormalized ratio), ascites, or hepatic encephalopathy. In otherembodiments, the DSI equation has one or more additional termsrepresenting a liver metabolic test result, where the test is selectedfrom caffeine elimination, antipyrine clearance, galactose eliminationcapacity, and formation of MEGX from lidocaine.

In some embodiments, the DSI can be used for defining disease severityin patients with chronic liver disease, tracking disease progression andresponse to treatments; wherein the chronic liver disease is selectedfrom chronic hepatitis C, primary sclerosing cholangitis (PSC),nonalcoholic fatty liver disease (NAFLD), chronic hepatitis B, alcoholicliver disease, autoimmune liver disease, cryptogenic cirrhosis,hemochromatosis, Wilson's disease, alpha-1-antitrypsin deficiency, andcholestatic liver diseases.

In some embodiments, DSI is used in a method for prediction of futureclinical outcomes. In some embodiments, the clinical outcomes areselected from Child-Turcotte-Pugh (CTP) increase, varices,encephalopathy, ascites, and liver related death. At the optimumcutoffs, DSI surprisingly outperformed other standard test methods forprediction of future clinical outcomes, as shown in Example 9.Specifically, Example 9 provides evidence that DSI exhibited the highestsensitivity, specificity, positive predictive value (PPV), and negativepredictive value (NPV) when compared to liver biopsy, platelet count andMELD.

In some embodiments, a DSI can be used for assessment of liver functionfor a number of specific clinical applications, for example, forprediction of response to an antiviral treatment in a patient with CHC,selection of patients with chronic hepatitis B who should receiveantiviral therapy; assessing the risk of hepatic decompensation inpatients with hepatocellular carcinoma (HCC) being evaluated for hepaticresection; identifying a subgroup of patients on waiting list with lowMELD (Model for End-stage Liver Disease score) who are at-risk for dyingwhile waiting for an organ donor; as an endpoint in clinical trials;replacing liver biopsy in pediatric populations; tracking of allograftfunction; measuring return of function in living donors; and measuringfunctional impairment in cholestatic liver disease (PSC, PrimarySclerosing Cholangitis).

In a specific embodiment, a DSI is used to predict the response totreatment, e.g., % of patients with CHC who will achieve SVR followingtreatment with PEG/RBV. In this specific embodiment, the DSI equation isDSI=9.84 (SHUNT)−12.36 LOGe (portal HFR)+50.5where SHUNT is SHUNT test value in the patient and portal HFR is portalHFR test value in the patient. Applying the DSI equation to the group ofpatients gave a cut-off of 30, with no one above this cutoff able toachieve SVR. Of the patients with DSI of 25-30 there were 15% thatachieved SVR. Of the patients with DSI of 20-25 there were 16% thatachieved SVR. Of the patients with DSI of less than 20 there were 19%that achieved SVR. See Table 11.

Distinguishable Compound. In some embodiments, portal flow can beassessed utilizing any safely orally administered distinguishablecompound with the following characteristics: 100% absorption followingoral administration, high hepatic extraction (>70% in first pass throughthe liver of a healthy subject), and removal from the blood or plasmaexclusively by the liver. The distinguishable compound for measurementof portal flow can be an endogenous compound or a xenobiotic.

In some embodiments, the distinguishable compound can be any labeledendogenous bile acid or bile acid conjugate; for example, thedistinguishable compound can be a distinguishable cholate compoundselected from any of the following labeled compounds: cholic acid, anyglycine conjugate of cholic acid, any taurine conjugate of cholic acid;chenodeoxycholic acid, any glycine conjugate of chenodeoxycholic acid,any taurine conjugate of chenodeoxycholic acid; deoxycholic acid, anyglycine conjugate of deoxycholic acid, any taurine conjugate ofdeoxycholic acid; or lithocholic acid, or any glycine conjugate ortaurine conjugate thereof. In various aspects, any bile acid or bileacid conjugate may be in the form of a physiologically acceptable salt,e.g., the sodium salt of cholic acid. In one aspect, the term cholicacid refers to the sodium salt of cholic acid. Cholic acid (cholate) isthe distinguishable cholate compound in some preferred embodiments. Asused herein, the terms cholate compound, cholate and cholic acid areused interchangeably.

Xenobiotics that could be administered orally and also have high firstpass hepatic elimination could include, but are not limited to,propranolol, nitroglycerin or derivative of nitroglycerin, or galactoseand related compounds.

In some embodiments, the distinguishable compound is propranolol.Propranolol is a nonselective β blocker and has been shown to beeffective for the prevention of variceal bleeding and rebleeding and iswidely used as the pharmacotherapy for the treatment of portalhypertension in patients with cirrhosis. (Suk et al. 2007, Effect ofpropranolol on portal pressure and systemic hemodynamics in patientswith liver cirrhosis and portal hypertension: a prospective study. Gutand Liver 1 (2): 159-164). Propranolol is almost entirely cleared by theliver. It has been demonstrated that total (+)-propranolol plasmaclearance constitutes a good estimate of hepatic blood flow in patientswith normal liver function. (Weiss et al., 1978 (+)-Propranololclearance, an estimation of hepatic blood flow in man, Br. J. Clin.Pharmacol. 5: 457-460).

In other embodiments, the distinguishable compound is isosorbide5-mononitrate. This compound can be administered orally and detected inplasma by HPLC-EIMS. (Sun et al., High performance liquidchromatography-electrospray ionization mass spectrometric determinationof isosorbide 5-mononitrate in human plasma, J. Chromatogr. B Analyt.Technol. Biomed. Sci. 2007 Feb. 1; 846(1-2):323-8).

In some embodiments, the distinguishable compound is galactose.Galactose elimination capacity (GEC) has been used as an index ofresidual hepatic function. Galactose in the GEC test typically isadministered intravenously at a dose of 0.5 mg/kg and venous samplestaken every 5 min between 20 and 60 minutes. The clearance of galactoseis decreased in individuals with chronic liver disease and cirrhosis.The fact that this carbohydrate has a high extraction ratio, however,makes the metabolism of galactose dependent on liver blood flow andhepatic functional mass. (Tygstrup N, Determination of the hepaticelimination capacity (Lm) of galactose by a single injection, Scand JLab Clin invest, 18 Suppl 92, 1966, 118-126). The carbohydrate galactoseis metabolized almost exclusively in the liver, and the elimination rateat blood concentrations high enough to yield near-saturated enzymaticconversion, the GEC is used as a quantitative measure of the metaboliccapacity of the liver. One study has shown that among patients with anewly-diagnosed cirrhosis and a decreased GEC, the GEC was a strongpredictor of mortality. (Jepsen et al, 2009, The galactose eliminationcapacity and mortality in 781 Danish patients with newly-diagnosed livercirrhosis: a cohort study. BMC Gastroenterol. 2009, 9:50).

In certain embodiments, one or more differentiable isotopes areincorporated into the selected distinguishable compound in order to beutilized to assess hepatic function. The differentiable isotope can beeither a radioactive or a stable isotope incorporated into the testcompound. Stable (¹³C, ²H, ¹⁵N, ¹⁸O) or radioactive isotopes (¹⁴C, ³H,Tc-99m) can be used. Advantages of stable isotopes are the lack ofexposure to radioactivity, natural abundance, and the specificity of theanalyses used for test compound identification (mass determination bymass spectrometry). Stable isotopically labeled compounds arecommercially available. For example, ¹³C- and ²H-labeled cholic acidcompounds can be purchased from Sigma-Aldrich, CDN Isotopes andCambridge Isotope Laboratories, Inc.

In some embodiments, the distinguishable compound for oraladministration can be any distinguishable cholate compound that isdistinguishable analytically from an endogenous cholic acid. In oneaspect, the distinguishable cholate compound is selected from anyisotopically labeled cholic acid compound known in the art.Distinguishable cholate compounds used in any one of these assays mightbe labeled with either stable (¹³C, ²H, ¹⁸O) or radioactive (¹⁴C, ³H)isotopes. Distinguishable cholate compounds can be purchased (forexample CDN Isotopes Inc., Quebec, CA). In a preferred aspect, thedistinguishable cholate is selected from any known safe, non-radioactivestable isotope of cholic acid. In one specific aspect, thedistinguishable cholate compound is 2,2,4,4-²H cholic acid. In anotherspecific aspect, the distinguishable cholate compound is 24-¹³C cholicacid.

In other embodiments, the distinguishable compound may be an unlabeledendogenous compound, such as unlabeled cholate. In the aspect using anunlabeled endogenous compound, the oral test dose is sufficiently great,for example 2.5-7.5 mg/kg cholate, for the resulting serum concentrationto be distinguishable above the baseline serum concentration of thatendogenous compound.

The platform for detecting and measuring the distinguishable compound inthe blood sample from the subject is dependent on the type ofadministered distinguishable compound. For stable isotopes, theconcentration of the distinguishable compound in a blood sample can bemeasured by, e.g. gas chromatography/mass spectroscopy (GC/MS) or liquidchromatography/mass spectroscopy (LC/MS). For radiolabeled testcompounds, e.g., scintillation spectroscopy can be employed. Foranalysis of unlabeled compounds, e.g., autoanalyzers, luminescence, orELISA can be employed. It is further contemplated that strip tests witha color developer sensitive directly or indirectly to the presence andquantity of test compound can be employed for use in a home test or apoint of care test.

Portal Blood Flow

Portal blood flow has been found to be the key to liver assessment. Theliver receives ˜75% of its blood through the portal vein which brings inthe nutrients for processing and deleterious compounds fordetoxification. This low blood pressure system is sensitive to theearliest disruption of the microvasculature so that the early stages ofCLD can be detected by decreased portal flow and increased shuntingbefore any other physiological impacts. The high pressure hepaticsystemic blood flow is decreased less and only later in the diseaseprocess. Unlike biopsy which samples only 1/50,000^(th) of the liver,the portal flow is a measure of the entire organ. As disease progressesthere is increasing disruption of the microvasculature architecture andincreasing impairment of portal flow which causes the majormanifestations of advanced CLD. Impaired flow causes ascites, portalhypertension, and esophageal varices. Impaired flow causes increasedshunting of toxins which leads to hepatic encephalopathy.

Cholate is a unique probe of the portal blood flow and the hepaticsystemic flow. Many liver tests have attempted to use the clearance oforal or IV compounds but only cholate has succeeded in assessing earlyand late stage CLD. Other oral compounds are absorbed at various sitesalong the GI tract and do not target the portal circulation. Othercompounds are taken up by nonspecific transporters. Oral cholate isspecifically absorbed by the terminal ileum epithelial cells via thehigh affinity ileal Na⁺-dependent bile salt transporter (ISBT) and iseffluxed by MRP3 transporters directly into the portal blood flow(Trauner and Boyer, 2003, Bile salt transporters: Molecularcharacterization, function, and regulation. Physiol Rev. 83: 633-671). Adifferent set of high affinity transporters including theNa⁺/taurocholate cotransporter (NTCP) and organic anion transportingproteins (OATPs) then takes it up into hepatocytes with highly efficientfirst pass extraction (Trauner and Boyer, 2003, infra) so that anycholate that escapes extraction is a direct measure of the portal flow.Once intracellular, it is rapidly conjugated to glycine and taurine sothat the unconjugated form does not then re-appear in the intrahepaticcirculation, which would confuse the pharmacokinetics. Otherunconjugated bile salts such as deoxycholate and chenodeoxycholate wouldbehave similarly but they are much stronger solubilizing agents andwould not be as safe to administer. Patient safety is ensured by using astable isotope labeled endogenous compound avoiding the risks ofxenobiotic or radiation exposure. All the proteins and systems involvedare highly conserved and essential so that the pharmacokinetics ofcholate are consistent between individuals and not affected by gender,age, or genetic makeup, or by diet or concomitant medications.

The portal blood flow can be non-invasively and accurately quantified byexploiting the unique physiology of the endogenous bile acid, cholate,which can be labeled, for example, with safe non-radioactive stableisotopes. Highly conserved enteric transporters (ISBT, MRP3)specifically target oral cholate to the portal circulation. Highlyconserved hepatic transporters (NTCP, OATPs) clear cholate from theportal and systemic circulation. Therefore, noninvasive quantitativeassessment of the portal circulation can be performed by administrationto a patient of a distinguishable cholate compound and assessment of alevel of the distinguishable cholate compound in blood samples drawn atvarious multiple time points to determine an oral clearance curve. TheFLOW (portal HFR) test accurately measures the portal blood flow from aminimum of 5 blood samples taken over a period of 90 minutes after anoral dose of deuterated-cholate.

A major study of almost 300 CHC patients, portal flow measured bycholate testing was superior in predicting clinical outcomes to thecurrent gold standard of fibrosis measured by biopsy (Everson et al,2011). In the Early CHC study impairment of the portal flow andincreased shunting measured by cholate testing was the earliestdetectable pathophysiology. These results have led to a newunderstanding of CLD that it is the disruption of hepaticmicrovasculature and not fibrosis per se that is deleterious. Thismicrovasculature disruption impairs the portal blood flow which can benon-invasively and accurately quantified by exploiting the uniquephysiology of the endogenous bile acid, cholate.

Portal-Systemic Shunting

As shown in FIG. 1, oral cholate is taken up by specific enterictransporters directly into the portal vein and removed by hepatictransporters in its first-pass through the liver. IV cholate distributessystemically and is extracted by both the hepatic artery and portalvein. In typical embodiments, concentrations of both oral and IVcholates are measured at 5 different times within 90 minutes ofadministration and clearances are calculated. The IV clearance over theoral clearance is the portal-systemic SHUNT fraction. The oral clearanceper kilogram of body weight represents the Portal Hepatic FiltrationRate (Portal HFR, FLOW), or amount of portal blood delivery. STAT is theconcentration of oral cholate at 60 minutes, and was shown to accuratelyestimate the portal HFR.

The SHUNT test non-invasively and accurately measures the portal bloodflow following oral administration of a distinguishable cholate compoundand also measures the systemic hepatic blood flow following intravenousco-administration of a second distinguishable cholate compound.Therefore the SHUNT test can be used to determine the amount ofportal-systemic shunting. In some embodiments, an IV dose of^(n)C-cholate is administered concurrently with an oral dose ofdeuterated-cholate and a minimum of 5 blood samples taken over a periodof 90 minutes after administration.

The dual cholate clearance SHUNT method yields 3 test results:Portal-systemic shunt fraction (SHUNT (%)); Portal Hepatic FiltrationRate (Portal HFR, which is also defined as FLOW in above discussions andexamples, (mL/min/kg)) based on orally administered distinguishablecholate compound in the blood; and Systemic Hepatic Filtration rate(Systemic HFR, (mL/min/kg)), based on intravenously administereddistinguishable cholate compound in the blood. Cholate-2,2,4,4-d4 (40mg) is given orally and taken up into the portal vein by specificenteric transporters. Cholate-24-¹³C (20 mg) is given IV and is taken upprimarily through the hepatic artery from the systemic circulation.Specific hepatic transporters clear cholate from the portal and systemiccirculation. For example, highly conserved hepatic transporters (NTCP,OATPs) clear cholate from the portal and systemic circulation.

Estimation of Portal Hepatic Filtration Rate

The STAT test is a simplified, non-invasive convenient test intended forscreening purposes can reasonably estimate the portal blood flow from asingle blood sample taken at a single time point, e.g., 60 minutes afteroral administration of a distinguishable cholate compound, e.g., adeuterated cholate.

Comparison of Portal HFR (FLOW), SHUNT and STAT Tests.

A comparison of typical embodiments of SHUNT, FLOW and STAT tests isshown in Table 1 below.

TABLE 1 Liver Function Tests. What is Test Test Route of Measured orName Compound Administration Samples Defined SHUNT ¹³C-cholateIntravenous n = 5 over Clearances and 4D-²H-cholate Oral 90 min Shunt-comprehensive assessment of hepatic blood flow and hepatic function FLOW4D-²H-cholate Oral n = 5 over Portal circulation 90 min (portal hepaticfiltration rate; Portal HFR) STAT 4D-²H-cholate Oral n = 1 at Estimates60 min FLOW and correlates with SHUNT

Values for normal liver function were established in healthy controls inprevious studies: the average SHUNT is 20%, average HFR (FLOW) is 30,and average STAT is 0.4.

In the diseased liver, as more blood escapes extraction by intra- andextra-hepatic shunting to the systemic circulation, the SHUNT increases(˜30-90%), HFR (FLOW) or portal flow decreases (˜20 to 2 mL/min/kg), andSTAT increases (0.6 to 5 uM).

Definitions and Acronyms

As used herein, “a” or “an” may mean one or more than one of an item.

The term “about” when referring to any numerical parameter means+/−10%of the numerical value. For example, the phrase “about 60 minutes”refers to 60 minutes+/−6 minutes.

As used herein “clearance” may mean the removing of a substance from oneplace to another.

As used herein the terms, “patient”, “subject” or “subjects” include butare not limited to humans, the term may also encompass other mammals, ordomestic or exotic animals, for example, dogs, cats, ferrets, rabbits,pigs, horses, cattle, birds, or reptiles.

The acronym “HALT-C” refers to the Hepatitis C Antiviral Long-termTreatment against Cirrhosis trial. The HALT-C trial was a large,prospective, randomized, controlled trial of long-term low dose peginterferon therapy in patients with advanced hepatitis C who had not hada sustained virologic response to a previous course of interferon-basedtherapy. An NIH-sponsored Hepatitis C Antiviral Long-Term Treatmentagainst Cirrhosis (HALT-C) Trial examined whether long-term use ofantiviral therapy (maintenance treatment) would slow the progression ofliver disease. In noncirrhotic patients who exhibited significantfibrosis, effective maintenance therapy was expected to slow or stophistological progression to cirrhosis as assessed by serial liverbiopsies. However, tracking disease progression with biopsy carries riskof complication, possibly death. In addition, sampling error andvariation of pathologic interpretation of liver biopsy limits theaccuracy of histologic assessment and endpoints. The histologic endpointis less reliable because advanced fibrosis already exists and changes infibrosis related to treatment or disease progression cannot be detected.Thus, standard endpoints for effective response to maintenance therapyin cirrhotic patients are prevention of clinical decompensation(ascites, variceal hemorrhage, and encephalopathy) and stabilization ofliver function as measured clinically by Childs-Turcotte-Pugh (CTP)score. However, clinical endpoints and CTP score were known to beinsensitive parameters of disease progression. Dual isotope techniquesemploying distinguishable cholates were used in development of the SHUNTtest and used in conjunction with the HALT-C trial.

The term “SHUNT test” refers to a previously disclosed QLFT(quantitative liver function test) used as a comprehensive assessment ofhepatic blood flow and liver function. The SHUNT test is used todetermine plasma clearance of orally and intravenously administeredcholic acid in subjects with and without chronic liver disease. In theSHUNT test, at least 5 blood samples are analyzed which have been drawnfrom a patient at intervals over a period of at least about 90 minutesafter oral and intravenous administration of differentiable cholates.Analysis of samples for stable isotopically labeled cholates isperformed by, e.g., GC-MS, following sample derivitization, or LC-MS,without sample derivitization. The ratio of the AUCs of orally tointravenously administered cholic acid, corrected for administereddoses, defines cholate shunt. The cholate shunt can be calculated usingthe formula: AUC_(oral)/AUC_(iv)×Dose_(iv)/Dose_(oral)×100%, whereinAUC_(oral) is the area under the curve of the serum concentrations ofthe orally adminstered cholic acid and AUC_(iv) is the area under thecurve of the intravenously administered cholic acid. The SHUNT test isdisclosed in Everson et al., US2008/0279766, Methods for Diagnosis andIntervention of Hepatic Disorders, filed Jan. 26, 2006, which isincorporated herein by reference. These studies demonstrated reducedclearance of cholate in patients who had either hepatocellular damage orportosystemic shunting. The “SHUNT test value” refers to a number (in%).

The SHUNT test allows measurement of first-pass hepatic elimination ofbile acids from the portal circulation. Flow-dependent, first passelimination of bile acids by the liver ranges from 60% for unconjugateddihydroxy, bile acids to 95% for glycine-conjugated cholate. Freecholate, used herein has a reported first-pass elimination ofapproximately 80% which agrees closely with previously observed firstpass elimination in healthy controls of about 83%. After uptake by theliver, cholic acid is efficiently conjugated to either glycine ortaurine and secreted into bile. Physicochemically cholic acid is easilyseparated from other bile acids and bile acid or cholic acid conjugates,using chromatographic methods.

The acronym “IV” or “iv” refers to intravenous.

The term “sustained virologic response” (SVR) is used to describe adesired response in a patient when hepatitis C virus is undetectable inthe blood six months after finishing treatment. Conventional treatmentusing interferon and ribavirin doesn't necessarily eliminate, or clear,the hepatitis C virus. A sustained virologic response is associated witha very low incidence of relapse. SVR is used to evaluate new medicinesand compare them with proven therapies.

The acronym “PO” refers to per oral.

The acronym “PHM” refers to perfused hepatic mass.

The acronym “SF” refers to shunt fraction, for example, as in cholateSF.

The acronym “ROC” refers to receiver operating characteristic. The ROCcurve is a graphical plot which illustrates performance of a binaryclassifier system as its discrimination threshold is varied. It iscreated by plotting the fraction of true positives out of the positives(TPR=true positive rate) vs. the fraction of false positives out of thenegatives (FPR=false positive rate), at various threshold settings.Sensitivity is the probability of a positive test result, or of a valueabove a threshold, among those with disease. Sensitivity is defined asthe true positive rate (TPR): TPR=TP/P=TP/(TP+FN). False positive rate(FPR) is FPR=FP/N=FP/(FP+FN). Accuracy (ACC) is defined asACC=(TP+TN)/(P+N). Specificity is the probability of a negative testresult, or a value below a threshold, among those without disease.Specificity (SPC), or true negative rate (TN) is defined asSPC=TN/N=TN/(FP+TN)=1−FPR. Positive prediction value (PPV) is definedas: PPV=TP/(TP+FP). Negative predictive value (NPV) is defined asNPV=TN/(TN+FN). The c-statistic is the area under the ROC curve, or“AUROC” (area under receiver operating characteristic curve) and rangesfrom 0.5 (no discrimination) to a theoretical maximum of 1 (perfectdiscrimination).

The term “oral cholate clearance” (Cl_(oral)) refers to clearance fromthe body of a subject of an orally administered cholate compound asmeasured by a blood or serum sample from the subject. Oral cholateclearance is used as a measure of portal blood flow. Orally administeredcholic acid is absorbed across the epithelial lining cells of the smallintestine, bound to albumin in the portal blood, and transported to theliver via the portal vein. Approximately 80% of cholic acid is extractedfrom the portal blood in its first pass through the liver. Cholic acidthat escapes hepatic extraction exits the liver via hepatic veins thatdrain into the vena cava back to the heart, and is delivered to thesystemic circulation. The area under the curve (AUC) of peripheralvenous concentration versus time after oral administration of cholicacid quantifies the fraction of cholic acid escaping hepatic extractionand defines “oral cholate clearance”.

The term “portal hepatic filtration rate”, “portal HFR”, “FLOW test”refers to oral cholate clearance (portal hepatic filtration rate; portalHFR) used as a measure of portal blood flow, or portal circulation,obtained from analysis of concentration of distinguishable cholatecompound in at least 5 blood samples drawn from a subject over a periodof, for example, about 90 minutes after oral administration of adistinguishable cholate compound, for example, a distinguishablecholate. The units of portal HFR value are typically expressed asmL/min/kg, where kg refers to kg body weight of the subject.

The term “STAT test” refers to an estimate of portal blood flow byanalysis from one patient blood sample drawn at a defined period of timefollowing oral administration of a differentiable cholate. In oneaspect, the STAT test refers to analysis of a single blood sample drawnat a specific time point after oral administration of a differentiablecholate. In one specific aspect, the STAT test is a simplifiedconvenient test intended for screening purposes that can reasonablyestimate the portal blood flow (estimated flow rate) from a single bloodsample taken 60 minutes after orally administered deuterated-cholate.The STAT test value is typically expressed as a concentration, forexample, micromolar (uM) concentration.

The term “intravenous cholate clearance” (Cl_(iv)) refers to clearanceof an intravenously administered cholate compound. Intravenouslyadministered cholic acid, bound to albumin, distributes systemically andis delivered to the liver via both portal venous and hepatic arterialblood flow. The AUC of peripheral venous concentration versus time afterintravenous administration of cholic acid is equivalent to 100% systemicdelivery of cholic acid. The ratio of the AUCs of orally tointravenously administered cholic acid, corrected for administereddoses, defines cholate shunt.

The term “Quantitative Liver Function Test” (QLFT), refers to assaysthat measure the liver's ability to metabolize or extract testcompounds, can identify patients with impaired hepatic function atearlier stages of disease, and possibly define risk for cirrhosis,splenomegaly, and varices. One of these assays is the cholate shuntassay where the clearance of cholate is assessed by analyzing bodilyfluid samples after exogenous cholate has been taken up by the body.

The term “Ishak Fibrosis Score” is used in reference to a scoring systemthat measures the degree of fibrosis (scarring) of the liver, which iscaused by chronic necroinflammation. A score of 0 represents nofibrosis, and 6 is established fibrosis. Scores of 1 and 2 indicate milddegrees of portal fibrosis; stages 3 and 4 indicate moderate (bridging)fibrosis. A score of 5 indicates nodular formation and incompletecirrhosis, and 6 is definite cirrhosis.

The term “Childs-Turcotte-Pugh (CTP) score” or “Child-Pugh score” refersto a classification system used to assess the prognosis of chronic liverdisease as provided in Pugh et al., Transection of the oesophagus forbleeding oesophageal varices. Br J Surg 1973; 60:646-649, which isincorporated herein by reference. The CTP score includes five clinicalmeasures of liver disease; each measure is scored 1-3, with 3 being themost severe derangement. The five scores are added to determine the CTPscore. The five clinical measures include total bilirubin, serumalbumin, prothrombin time international normalized ratio (PT INR),ascites, and hepatic encephalopathy. The CTP score is one scoring systemused in stratifying the seriousness of end-stage liver disease. Chronicliver disease is classified into Child-Pugh class A to C, employing theadded score. Child-Pugh class A refers to CTP score of 5-6. Child-Pughclass B refers to CTP score of 7-9. Child-Pugh class C refers to CTPscore of 10-15. A website calculates post-operative mortality risk inpatients with cirrhosis. mayoclinic.org/meld/mayomodel9.html

The term “Model for End-Stage Liver Disease (MELD) refers to a scoringsystem used to assess the severity of chronic liver disease. MELD wasdeveloped to predict death within three months of surgery in patientswho had undergone a transjugular intrahepatic portosystemic shunt (TIPS)procedure patients for liver transplantation. MELD is also used todetermine prognosis and prioritizing for receipt of a liver transplant.The MELD uses a patient's values for serum bilirubin, serum creatinine,and international normalized ratio for prothrombin time (INR) to predictsurvival. The scoring system is used by the United Network for OrganSharing (UNOS) and Eurotransplant for prioritizing allocation of livertransplants instead of the older Child-Pugh score. See UNOS (2009 Jan.28) “MELD/PELD calculator documentation”, which is incorporated hereinby reference. For example, in interpreting the MELD score inhospitalized patients, the 3 month mortality is: 71.3% mortality for aMELD score of 40 or more

The term “standard sample” refers to a sample with a known concentrationof an analyte used for comparative purposes when analyzing a samplecontaining an unknown concentration of analyte.

The term “Chronic Hepatitis C” (CHC) refers to a chronic liver diseasecaused by viral infection and resulting in liver inflammation, damage tothe liver and cirrhosis. Hepatitis C is an infection caused by ablood-borne virus that attacks the liver and leads to inflammation. Manypeople infected with hepatitis C virus (HCV) do not exhibit symptomsuntil liver damage appears, sometimes years later, during routinemedical tests.

The term “Alcoholic SteatoHepatitis” (ASH) refers to a chronic conditionof inflammation of the liver which is caused by excessive drinking.Progressive inflammatory liver injury is associated with long-term heavyintake of ethanol and may progress to cirrhosis.

The term “Non-Alcoholic SteatoHepatitis” (NASH) refers to a seriouschronic condition of liver inflammation, progressive from the lessserious simple fatty liver condition called steatosis. Simple steatosis(alcoholic fatty liver) is an early and reversible consequence ofexcessive alcohol consumption. However, in certain cases the fataccumulation can be associated with inflammation and scarring in theliver. This more serious form of the disease is termed non-alcoholicsteatohepatitis (NASH). NASH is associated with a much higher risk ofliver fibrosis and cirrhosis than NAFLD. NAFLD may progress to NASH withfibrosis cirrhosis and hepatocellular carcinoma.

The term “Non-Alcoholic Fatty Liver Disease” (NAFLD) refers to a commonchronic liver disease characterized in part by a fatty liver conditionwith associated risk factors of obesity, metabolic syndrome, and insulinresistance. Both NAFLD and NASH are often associated with obesity,diabetes mellitus and asymptomatic elevations of serum ALT and gamma-GT.Ultrasound monitoring can suggest the presence of a fatty infiltrationof the liver; differentiation between NAFLD and NASH, typically requiresa liver biopsy.

The term “Primary Sclerosing Cholangitis” (PSC) refers to a chronicliver disease caused by progressive inflammation and scarring of thebile ducts of the liver. Scarring of the bile ducts can block the flowof bile, causing cholestosis. The inflammation can lead to livercirrhosis, liver failure and liver cancer. Chronic biliary obstructioncauses portal tract fibrosis and ultimately biliary cirrhosis and liverfailure. The definitive treatment is liver transplantation. Indicationsfor transplantation include recurrent bacterial cholangitis, jaundicerefractory to medical and endoscopic treatment, decompensated cirrhosisand complications of portal hypertension (PHTN). PSC progresses throughchronic inflammation, fibrosis/cirrhosis, altered portal circulation,portal hypertension and portal-systemic shunting to varices-ascites andencephalopathy. Altered portal flow is an indication of clinicalcomplications.

Other definitions are provided throughout the specification.

Computer/Processor

The detection, prognosis and/or diagnosis method employed in the STATtest can employ the use of a processor/computer system. For example, ageneral purpose computer system comprising a processor coupled toprogram memory storing computer program code to implement the method, toworking memory, and to interfaces such as a conventional computerscreen, keyboard, mouse, and printer, as well as other interfaces, suchas a network interface, and software interfaces including a databaseinterface find use one embodiment described herein.

The computer system accepts user input from a data input device, such asa keyboard, input data file, or network interface, or another system,such as the system interpreting, for example, the LC-MS or GC-MS data,and provides an output to an output device such as a printer, display,network interface, or data storage device. Input device, for example anetwork interface, receives an input comprising detection ofdistinguishable cholate compound measured from a processed blood orserum sample described herein and quantification of those compounds. Theoutput device provides an output such as a display, including one ormore numbers and/or a graph depicting the detection and/orquantification of the compounds.

Computer system is coupled to a data store which stores data generatedby the methods described herein. This data is stored for eachmeasurement and/or each subject; optionally a plurality of sets of eachof these data types is stored corresponding to each subject. One or morecomputers/processors may be used, for example, as a separate machine,for example, coupled to computer system over a network, or may comprisea separate or integrated program running on computer system. Whichevermethod is employed these systems receive data and provide data regardingdetection/diagnosis in return.

In embodiments, a method for selecting a treatment for a subject thathas an abnormal level of distinguishable cholate compound in a blood orserum sample drawn at a single time point following oral administrationcomprises calculating an output score, using a computing device, byinputting the distinguishable cholate compound level into a functionthat provides a predictive relationship between cholate level andoutcome, for subjects having a liver disease or disorder; and displayingthe output score, using a computing device.

In embodiments, a STAT test value is obtained following oraladministration of a distinguishable cholate compound to the subject, asingle blood or serum sample is drawn at a specific time point followingadministration. In some embodiments, the STAT test value, expressed asconcentration of distinguishable cholate compound in the sample isconverted by using an equation into an estimated portal flow rate, orestimated portal HFR (FLOW) (expressed as mL/min/kg) in the subject. Inembodiments, the equation is y=0.9702x+0.0206, where x is the logHepquant FLOW and y is LOG Hepquant STAT.

In embodiments, the method further comprises determining whether theoutput score is greater than, or equal to, or less than a cutoff value,using a computing device; and displaying whether the subject is likelyto experience a clinical outcome if the output score is greater than, orequal to, or less than a cutoff value.

In embodiments, a computing device, comprises a processing unit; and asystem memory connected to the processing unit, the system memoryincluding instructions that, when executed by the processing unit, causethe processing unit to: calculate a level of distinguishable cholatecompound from a single blood sample from a subject into a function thatprovides a predictive relationship between distinguishable cholate levelof the subject having a liver disease or dysfunction; and display theoutput score. In embodiments, the system memory includes instructionsthat when executed by the processing unit, cause the processing unit todetermine whether the output score is greater than or equal to or lessthan a cutoff value; and displaying whether the subject is likely toexperience a clinical outcome if the output score is greater than orequal to the cutoff value.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. The following examples are included to demonstratepreferred embodiments.

Example 1. STAT Test-Estimating Portal Flow from a Single Blood Draw

The individual time point serum cholate concentrations from the portalHFR (FLOW) and SHUNT tests in HALT-C and Early CHC studies werecarefully analyzed and differences at 45, 60, and 90 minutes were foundto be highly significant (p<0.005). The concentration at 60 minutes hadthe best correlation (r²=0.8) with the portal flow. An equation wasderived that could transform the concentration (uM) at 60 min into anestimated portal flow (mL/min/kg) with 85% accuracy of the 5 point FLOWmethod. The equation is y=0.9702x+0.0206, where x is the LOG Portal HFR(FLOW) and y is LOG STAT. In the STAT test, the patient drinks an oraldose of distinguishable cholate compound, e.g., deuterated-cholate, andgives a single blood sample after 1 hour. The accuracy of the STAT testrelative to the FLOW (Portal HFR) test is shown in FIG. 9.

Example 2. Efficacy of STAT (Estimated Portal Flow) in Detecting HepaticDysfunction

In the Early CHC study healthy controls had a portal flow of 34±14ml/min/kg (mean±SD). Hepatic dysfunction was defined as a portal flowmore than 1 SD below the control mean, a flow <20 ml/min/kg. In theearly CHC group, about ½ the patients exhibited hepatic dysfunction. Theestimated portal flows in the early CHC patients were calculated fromthe equation shown in FIG. 8 using their 60 min serum cholate level. Theestimated flow could detect hepatic dysfunction with a sensitivity of90%, a specificity of 85%, a positive predictive value (PPV) of 82%, anda negative predictive value (NPV) of 92%. These preliminary resultsdemonstrate that a single blood sample after an oral cholate dose couldbe used to detect hepatic dysfunction in early stage CLD.

Furthermore, in the Early CHC study we analyzed the potential impact ofSTAT if used as a screening test. Currently adults are screened forliver disease in the primary care setting by ALT. In our analysis of theEarly CHC study we found that addition of STAT to ALT could improvedetection of patients with chronic hepatitis C. In early stage patients,ALT was abnormal in only 34%, STAT was abnormal in 48%, and 65% of thepatients had either abnormal ALT or STAT. Screening with combination ofALT and STAT would double the detection rate for patients with liverdisease due to chronic hepatitis C. Of course, when used in such astrategy, STAT would also detect patients with liver diseases other thanchronic hepatitis C as well.

STAT also has test cutoffs that correlate with advanced liver disease.In patients with chronic hepatitis C and in patients with the chroniccholestatic liver disease, primary sclerosing cholangitis, STAT resultwith estimated FLOW of <10 mL/(kg min) correlated with risk for liverdecompensation or clinical complications. In this situation, STAT wouldreflex to either FLOW or SHUNT to provide precise quantification of theportal circulation.

Example 3. Procedure for Performance of an Exemplary STAT Test

Supplies

PO (Per Oral) Test Compounds:

²H4-Cholate ([2,2,4,4-²H]-Cholic Acid, 40 mg) (e.g. CDN Isotopes).

Sodium bicarbonate (e.g. 600 mg).

Patient Testing Supplies:

Serum/plasma transfer tubes and labels.

10 cc syringe for drawing blood sample.

7 cc red top and 7 cc gray top vacutainer tubes for serum samplecollection.

Needle discard bucket

A drinking substance such as apple or grape juice for diluting oral testcompounds.

Exemplary Test Compound Preparation

One exemplary solution of an oral composition may contain2,2,4,4-²H-Cholate, and Sodium bicarbonate (e.g. 40 mg, and 600 mg,respectively). In one exemplary method, the day before the test, watercan be added to about the 10 cc mark on a tube containing the oral testcompounds to obtain the Oral Test Solution. Cap tube tightly and shaketo mix. Swirl contents to get all the powder granules down into thewater.

On the test day pour dissolved Oral Test Solution into a container suchas a urine cup. Rinse tube into urine cup with about 10 mls water. Priorto beginning the test, add a diluting liquid such as grape or applejuice (not citrus juice) to about the 40 ml mark on the urine cupcontaining the Oral Test Solution. Swirl gently to mix; do not shake orstir, or mixture may foam out of container. Have extra juice on hand forrinse.

Testing Procedure

In one exemplary method the following procedure will be used. Optionallycollect baseline serum sample (see Sample Collection) before testcompound is administered.

Administration of Test Compounds.

Start timer. Record T=0.0—have patient drink oral solution of cholateand juice. Rinse cup with a little more juice and have patient drinkrinse. Record timer time.

Sample Collection

Blood

Collect the intravenous blood sample from the patient at 60 minutes postcholate administration. Record timer time.

Process blood samples and perform sample analysis by HPLC/MS (asoutlined below for FLOW and SHUNT); or by GC/MS to determine theconcentration of distinguishable cholate in the blood sample. The sampletest result for a given patient at a specific date/time point can becompared to cutoff values established from, e.g., a control group, oralternatively each patient may serve as his/her own control over time.

Example 4. Procedure for Performance of SHUNT and Portal HFR (FLOW)Assays with Analysis by HPLC-MS

Performance of Portal HFR (FLOW; Oral Cholate Clearance Test) and SHUNT(Cholate Shunt Test) assays are disclosed in US 2010/0055734 and US2008/0279766, each of which is incorporated herein by reference.

Clinical Protocol. The deuterated-cholate (product#614149) and¹³C-cholate (product#605883) are purchased from Sigma-Isotec (SaintLouis, Mo.) and dissolved in sodium bicarbonate buffer. The inventor hasheld the INDs #65121 and 65123 on these compounds since 2002 and reportsannually to the FDA. The ¹³C-cholate for injection is filtered, testedfor sterility and pyrogens, and frozen in aliquots by a researchpharmacist. After an overnight fast, each subject receives an indwellingintravenous catheter and a baseline venous blood sample is drawn. Thesubject drank the deuterated-cholate dose mixed with grape juice, and atthe same time, the ¹³C-cholate mixed with albumin is administered IV. Attime points of 5, 20, 45, 60, and 90 minutes, venous blood samples aredrawn. After processing to serum, samples are transferred to theClinical Testing Laboratory.

Laboratory Analyses. Patient serum samples are spiked with unlabeledcholate as internal standard and then the cholates are isolated by SPEand ether extraction. LCMS on C8 and Selected Ion Monitoring (SIM) areused to quantify the test compounds by the isotope dilution method. Allanalytical runs include appropriate standard curves and QC samples. Theoral clearance (FLOW test result) and IV clearance are calculated fromthe serum concentrations at the 5 time points. The ratio of IV to oralclearance is the SHUNT test result. The oral clearance is estimated fromonly the 60 minute time point and used as the STAT test result.

Detailed Procedures are Provided Below.

Collection and Processing of Samples.

Reagents and Supplies.

The following reagents and supplies are utilized in the Cholate Shuntand Cholate Clearance Test procedures. If the patient is undergoing onlythe oral cholate clearance test, the IV Solution and 25% Human Albuminfor injection are omitted.

IV Solution—20 mg 24-¹³C-Cholic Acid in 5 cc 1 mEq/ml Sodium Bicarbonate

PO test compounds 2,2,4,4-²H (40 mg) and Sodium Bicarbonate (600 mg)

25% Human Albumin for injection (5 ml) to be added to 24-¹³C-Cholic Acidsolution.

IV supplies, including 250 mls NS, indwelling catheter, 3-way stopcock.

10 cc syringes for administering IV test compounds

7 cc red top tubes for sample collection

3 ml crovials for serum storage

Needle discard bucket

Apple or Grape (non-citrus) juice for oral test compounds

Timer

Centrifuge

Transfer pipets

Patient preparation.

It is ascertained that the patient has no allergic reaction to latex. Itis further ascertained that the patient has had nothing to eat or drink(NPO), except water, since midnight the night before the test day. Thepatient height and weight are measured and recorded. The patient isfitted with an IV with a three-way stopcock and normal saline to keepopen (NS TKO) is placed before the test begins.

Cholate Compound Stock Solutions.

Test Compound Preparation.

The Oral Solution is utilized for either or both of the oral cholateclearance test and/or the cholate shunt assay. The oral solutionincluding 2,2,4,4-²H-Cholic acid (40 mg) and Sodium Bicarbonate (600 mg)is dissolved in about 10 cc water 24 hours prior to testing by mixingvigorously. The solution is stored in either the refrigerator or at roomtemperature. Just prior to administration, grape or apple (non-citrus)juice is added to the mixture. The juice solution is mixed well andpoured into cup for patient to drink. The cup is rinsed with extra juicewhich is administered to the patient.

The IV Solution is utilized for either or both of the IV cholateclearance test and/or the cholate shunt assay. A formulation of 20 mgCholic Acid-24-¹³C in 5 cc 1 mEq/ml Sodium Bicarbonate is prepared bypharmacy staff. The Test dose is 20 mg Cholic Acid-24-¹³C in 10 ccdiluent. If vial is frozen, it is allowed to thaw completely. Just priorto beginning the test, the Cholic Acid-24-¹³C solution is mixed withalbumin as follows (this method prevents loss of test compound duringmixing process). Draw up all of 24-¹³C-Cholic Acid solution (about 5 cc)in a 10 cc syringe. Draw up 5 cc albumin in another 10 cc syringe.Detach needle from the 24-¹³C-cholate syringe and attach a 3-waystopcock. Detach needle from albumin syringe and inject albumin throughstopcock into 24-¹³C Cholate syringe. Draw a little air into the bileacid/albumin syringe and mix solutions gently by inverting syringeseveral times. Expel air.

Test Compound Administration.

Collect baseline samples before test compounds are given. The time thesespecimens are collected should be recorded on sample collection recordsheet. Administration of test compounds is performed as follows. Starttimer. Record 24 hour clock time as T=0. Record time. At T=1-3 minutesadminister oral compounds. Have the patient drink the oral solution andjuice. Rinse cup with more juice and have patient drink rinse. Recordtimer time. At T=4-5 minutes-using the 3-way stopcock administer the IVpush of 20 mgs ¹³C Cholic acid in 5 mls 25% Human Albumin. Record timertime. Return line to NS through 3-way stopcock.

Specimen Collection.

Collect all samples via the 3-way stopcock with 0.5 ml discard beforeeach sample to prevent dilution or cross-contamination of samples.Collect 5 ml red tops at the following times. (T=timer time).

-   -   a. T=10 minutes, collect 5 minute, record timer time;    -   b. T=25 minutes, collect 20 minute, record timer time;    -   c. T=50 minutes, collect 45 minute, record timer time;    -   d. T=65 minutes, collect 60 minute, record timer time;    -   e. T=95 minutes, collect 90 minute, record timer time.        Specimen Handling.

Red top tubes are allowed to clot at room temperature for at least 30minutes. All blood tubes are spun for 10 minutes at 3000 rpm. Serum isremoved to properly labeled vials and frozen at −20° C. until samplesare transported.

Preparation of Cholate Compound Stock Solutions.

Accurate determination of cholate clearances and shunt is dependent onaccurate calibration standards. Concentrations of cholic acid compoundsin stock solutions must be accurate and reproducible. Very accurate(error <0.5%) portions of the cholic acid powders are weighed and glassweighing funnels and washes of 1 M NaHCO₃ are used to ensurequantitative transfer of the powder to the flask. Volumetric flasks areused to ensure accurate volumes so that the final concentrations of theprimary stock solutions are accurate. Calibrated air displacementpipettes are used to dispense accurate volumes of the primary stocksolutions that are brought to full volume in volumetric flasks toprepare secondary stock solutions that are also very accurate. Secondarystock solutions are used to prepare the standard curve samples, accuracyand precision samples, recovery samples, quality control samples,selectivity samples, and stability samples as described in theappropriate SOPs.

The following reagents are required.

1 M NaHCO₃

0.1 M NaHCO₃

0.1 M NaHCO₃/2% BSA

Methanol, LCMS grade

Water, CLRW grade (Clinical Laboratory Reagent Water)

Cholic Acid, purity 98%

Chenodeoxycholic Acid, purity 98%

[24-¹³C]-Cholic Acid, 99 atom % ¹³C

[2,2,4,4-²H]-Cholic Acid, 98 atom % ²H.

All primary stock solutions are prepared at a concentration of 250 uMusing Table 2 below.

TABLE 2 Cholate compound primary stock solutions. 13-C cholic 4-D cholicchenodeoxcholic cholic acid acid acid acid MW 408.56 409.59 412.60392.56 purity 98.0% 99.0% 98.0% 98.0% volume 100 ml 100 ml 100 ml 100 mlconc 250 uM 250 uM 250 uM 250 uM weight 10.42 mg 10.34 mg 10.53 mg 10.01mg

Primary stock solutions are prepared separately in 0.1 M NaHCO₃ and inmethanol as follows. Weigh out the appropriate amount of cholic acidcompound (+/−0.05 mg) in a glass weighing funnel. Transfer the powder toa 100 ml volumetric flask. Use either methanol or 0.1M NaHCO₃ to rinseany residual powder from the funnel into the flask. Bring to a finalvolume of 100 ml with methanol and mix well. Label flask with anexpiration of 1 month. Store at −20° C.

The unlabeled cholic acid is prepared as a 50 uM internal standard ineither MeOH or 0.1 M NaHCO₃ as follows. Pipette 2.0 ml of theappropriate 250 uM CA primary standard into a 10 ml volumetric flask.Bring to a total volume of 10 ml with 0.1 M NaHCO₃ or methanol and mixwell. Label flask with an expiration of 1 year. Store at 4° C.

[24-¹³C]-Cholic Acid secondary stock solutions made in methanol areshown in Table 3. Each secondary stock solution into the appropriate 15ml glass screw top test tube. Tubes are securely capped and sealed withseveral layers of parafilm and stored at −20° C.

TABLE 3 [24-¹³C]-Cholic acid secondary stock solutions in methanol.final assay Secondary 250 uM concentration Stocks 13C-CA (m) MethanolTotal uM uM ul ml ml 0.20 B (m) 2.0 80 + 9.92 = 10.00 1.00 D (m) 10.0400 + 9.60 = 10.00 6.00 F (m) 60.0 2400 + 7.60 = 10.00 2880 27.12 30.00

[2,2,4,4-²H]-Cholic Acid secondary stock solutions made in methanol areshown in Table 4. Each secondary stock solution into the appropriate 15ml glass screw top test tube. Tubes are securely capped and sealed withseveral layers of parafilm and stored at −20° C.

TABLE 4 [2,2,4,4-²H]-Cholic acid secondary stock solutions in methanol.final assay Secondary 250 uM concentration Stocks 4D-CA (m) MethanolTotal uM uM ul ml ml 0.30 I (m) 3.0 120 + 9.88 = 10.00 1.00 K (m) 10.0400 + 9.60 = 10.00 3.00 L (m) 30.0 1200 + 8.80 = 10.00 1720 28.28 30.00

[24-¹³C]-Cholic Acid secondary stock solutions made in 0.1 M NaHCO₃ andBSA are shown in Table 5. Each secondary stock solution is transferredinto the appropriate 15 ml screw top plastic tube, capped, sealed withseveral layers of parafilm and stored at 4° C.

TABLE 5 [24-¹³C]-Cholic acid secondary stock solutions in 0.1M NaHCO₃and BSA. final assay concen- Secondary 250 uM 0.1M 2% tration Stocks13C-CA NaHCO3 BSA Total uM uM ul ml ml ml 0.10 A 1.0 40 + 4.96 + 5.00 =10.00 0.20 B 2.0 80 + 4.92 + 5.00 = 10.00 0.60 C 6.0 240 + 4.76 + 5.00 =10.00 1.00 D 10.0 400 + 4.60 + 5.00 = 10.00 2.00 E 20.0 800 + 4.20 +5.00 = 10.00 6.00 F 60.0 2400 + 2.60 + 5.00 = 10.00 10.00 G 100.0 4000 +1.00 + 5.00 = 10.00 7960 27.04 35.00 70.00

[2,2,4,4-²H]-Cholic Acid secondary stock solutions made in 0.1 M NaHCO₃and BSA are shown in Table 6. Each secondary stock solution istransferred into the appropriate 15 ml screw top plastic tube, capped,sealed with several layers of parafilm and stored at 4° C.

TABLE 6 [2,2,4,4-²H]-Cholic acid secondary stock solutions in 0.1MNaHCO₃ and BSA. final assay concen- Secondary 250 uM 0.1M 2% trationStocks 4D-CA NaHCO3 BSA Total uM uM ul ml ml ml 0.10 H 1.0 40 + 4.96 +5.00 = 10.00 0.30 I 3.0 120 + 4.88 + 5.00 = 10.00 0.50 J 5.0 200 +4.80 + 5.00 = 10.00 1.00 K 10.0 400 + 4.60 + 5.00 = 10.00 3.00 L 30.01200 + 3.80 + 5.00 = 10.00 5.00 M 50.0 2000 + 3.00 + 5.00 = 10.00 396026.04 30.00 60.00

The secondary stock solutions as prepared above are utilized inpreparation of accuracy and precision samples in human serum withunlabeled cholate as an internal standard. The secondary stock solutionsare used in preparation of recovery samples with addition of unlabeledcholate as an internal standard.

In order to accurately measure patient liver function with the cholateshunt assay, the two different stable isotope cholate compounds musteach be accurately quantified in patient serum. In order to do this, theaccuracy, precision, and recovery of each of the two standard curvesmust be validated over their respective ranges of concentrations.

The accuracy and precision of an assay are assessed by running multiplereplica samples at the lower limit of quantification (LLOQ), low,medium, and high range of concentrations. Accuracy is the closeness ofthe average measured value to the actual value. Precision is thereproducibility of the measured value as indicated by the CV. Therecovery is assessed by comparing the detector response of the analyteextracted from serum relative to that of pure analyte measured at low,medium, and high concentrations.

Preparation of Quality Control Samples

The FDA provides guidance as to acceptable levels of accuracy andprecision of analytical methods. See, for example, Bioanalytical MethodValidation, May 2001, Section VI. Application of Validated Method toRoutine Drug Analysis. Once the analytical method has been validated forroutine use, its accuracy and precision should be monitored regularly toensure that the method continues to perform satisfactorily. To achievethis objective, a number of QC samples are prepared separately andshould be analyzed with processed test samples at intervals based on thetotal number of samples. The QC samples are run in duplicate at threeconcentrations (one near the lower limit of quantification (LLOQ) (i.e.,3×LLOQ), one in midrange, and one close to the high end of the range)and should be incorporated in each assay run. The number of QC samples(in multiples of three) will depend on the total number of samples inthe run. The results of the QC samples provide the basis of accepting orrejecting the run. At least four of every six QC samples should bewithin 15% of their respective nominal value. Two of the six QC samplesmay be outside the 15% of their respective nominal value, but not bothat the same concentration.

The QC samples must cover the high, middle, and low ranges of bothstandard curves. The QC samples are designed to closely simulate theactual concentrations of labeled compounds found in patient serum overthe time course of the testing. The [24-¹³C]-CA concentration is veryhigh at the early time point and falls exponentially to medium and lowconcentrations. The [2,2,4,4-²H]-CA concentration is very low at theearly time point, rises to its highest value in the middle time pointsand then falls to a medium concentration.

Supplies

The following supplies are utilized to prepare the QC samples used inthe Cholate Shunt and Cholate Clearance Test procedures. If the patientsamples are undergoing only the oral cholate clearance test, the[24-¹³C]-CA QC samples can be omitted.

Human Serum AB (Gemini Bio-Products #100-512)

Unlabeled Cholate Internal Standard Stock Solution (IS; 50 uM CholicAcid in 0.1M NaHCO₃)

[24-¹³C]-Cholic Acid and [2,2,4,4-²H]-Cholic Acid Secondary StockSolutions in 0.1 M NaHCO₃/1% BSA:

B 2.0 uM [24-¹³C]-CA

D 10.0 uM [24-¹³C]-CA

F 60.0 uM [24-¹³C]-CA

I 3.0 uM [2,2,4,4-²H]-CA

K 10.0 uM [2,2,4,4-²H]-CA

L 30.0 uM [2,2,4,4-²H]-CA

10 ml volumetric flasks

P1000 air displacement pipette and 1 ml tips

New, clean cryovials

Procedure for Preparation of Quality Control Samples for CholateClearance and Assays.

The [24-¹³C]-Cholic Acid and [2,2,4,4-²H]-Cholic acid QC samples areprepared as follows. For each set of QC samples, label 3 clean 10 mlvolumetric flasks as “QC 1”, “QC 2”, and “QC 3” as shown in Table 7.Larger volumetric flasks can be used to prepare larger batches. Use 1/10the nominal volume of the larger flasks as the amount of secondary stocksolution to add as indicated below.

TABLE 7 QC samples. Tubes [24-¹³C]-CA [2,2,4,4-²H]-CA QC1 1.00 ml F 1.00ml I QC2 1.00 ml D 1.00 ml L QC3 1.00 ml B 1.00 ml K

Using a P1000, add 1.0 ml of the appropriate [24-¹³C]-CA stock solutionand 1.0 ml of the appropriate [2,2,4,4-²H]-CA stock solution to theappropriate flasks as indicated in Table 6. Bring each flask to an exacttotal of 10.0 ml with human serum. Securely cap each flask and mix wellby inversion several times. Label 8 cryovials as “QC 1”, 8 as “QC 2”,and 8 as “QC 3”. Aliquot 1.2 ml of each QC mixture into the appropriatevials. Store the QC samples frozen at −80° C. QC samples have anexpiration of 1 year.

High Pressure Liquid Chromatography-Mass Spectroscopy (HPLC-MS) SamplePreparation

In order to ensure accurate liver function testing, the labeled cholatetest compounds must be isolated and identified from patients' serumsamples. Cholate compounds are amphipathic molecules with bothhydrophobic and hydrophilic regions. Cholates are also carboxylic acidsthat can exist in either an uncharged free acid form (cholic acid) or acharged carboxylic acid form (cholate) depending on pH. These propertiescan be exploited to isolate cholate compounds from serum. The use ofHPLC/MS as opposed to GC/MS, allows analysis of cholate without samplederivitization. Alternatively, GC/MS can be used for sample analysiswith derivitization by any technique known in the art, for example, bythe method of Everson and Martucci, US 2008/0279766, incorporated hereinby reference.

Reagents, Supplies and Equipment

The following reagents are prepared and used in the HPLC-MS samplepreparation.

Water, CLRW grade (Clinical Laboratory Reagent Water)

Methanol, LCMS grade

Diethyl Ether, ACS grade

Unlabeled Cholic Acid Internal Standard (IS) Primary Stock Solution (50uM CA in 0.1 M NaHCO₃)

Quality Control Samples (prepared as described above)

1.0 N NaOH (dissolve 20 g NaOH in 500 ml water)

0.01 N NaOH (dilute 1.0 N NaOH 1 to 100 with water)

10% Methanol (add 100 ml Methanol to a 1 L cylinder and bring to 1.0 Lwith water)

90% Methanol (add 900 ml Methanol to a 1 L cylinder and bring to 1.0 Lwith water)

0.2 N HCl (add 1.0 ml ACS grade Concentrated HCl slowly with stirring to57.0 ml water)

Mobile Phase (10 mM Ammonium Acetate/60% Methanol)

Disposable 16×100 and 13×100 test tubes

P1000 air displacement pipette and 1 ml tips

P100 air displacement pipette and 0.2 ml tips

Repeater Pipette

Vortex Mixer

SPE cartridges (Bond Elut LRC C18 OH, 500 mg, Varian, Inc)

Vacuum Manifold

Speed-Vac

Benchtop centrifuge

Speed-Vac vented to fume hood

Bath Sonicator

Repeater Dispensers for water, methanol, 10% methanol, and 90% methanol

Remove patient serum samples and a set of QC samples (2 each of QC1, 2,and 3) from the freezer and allow them to thaw to room temperature.Personal protective equipment (PPE) including lab coat, gloves, eyeprotection must be worn. All eluates and equipment must be disinfected.Pipettes and tips that come in contact with the sample must be discardedinto hazardous waste.

Label a set of test tubes (16×100) for each patient with that patient'sinitials and the time point code (5 min is 1, 20 min is 2, 45 min is 3,60 min is 4, 90 min is 5). Using a P1000 pipette, transfer 0.50 ml ofpatient's serum from the appropriate collection tube into theappropriate test tube.

Label a set of test tubes (16×100) for each QC sample (QC1a, QC1b, QC2a,QC2b, QC3a, QC3b). Using a P1000, transfer 0.50 ml of each QC sampleinto the appropriate test tube.

Label 2 test tubes (13×100) as STD1 and STD2.

To each patient sample and each QC sample and each STD sample tube, add50 ul of the Unlabeled Cholic Acid Internal Standard (IS) Primary StockSolution using a Repeater Pipette.

Set aside the STD tubes for later acidification and ether extraction instep 21.

To each patient sample tube and QC sample tube add 1.0 ml of 0.01 N NaOHwith a Repeater pipet and vortex 30 sec.

Label a set of SPE cartridges with one for each patient serum and QCsample to be processed.

In the hood add 5 ml Methanol with a repeater dispenser to eachcartridge. This step may be done on a vacuum manifold with high vacuumor by gravity. This wets the resin bed with solvent. Once the top of theliquid reaches the top of the frit add the next solution. Avoid lettingthe cartridges run dry.

Add 10 ml Water with the repeater dispenser to each cartridge. Thisequilibrates the resin bed to prepare it for binding cholate compounds.This step may be done on the vacuum manifold on high vacuum or bygravity.

To each SPE cartridge add the appropriate sample. The cholate compoundswill bind to the resin bed. To each sample test tube add a 1 ml waterrinse with the repeater, vortex, and add this rinse to the appropriatecartridge. Allow the sample to run by gravity for 20 minutes or longerthen may use low vacuum ≤3 inches Hg to pull sample through.

After the sample has completely entered the resin bed, add 2.5 ml Waterto each SPE cartridge with the repeater dispenser. This washes thecolumn resin bed. Use low vacuum ≤3 inches Hg.

To each SPE cartridge add 2.5 ml 10% Methanol with the repeaterdispenser. This further washes the column resin bed. Use low vacuum ≤3inches Hg.

Label a set of test tubes (13×100) with one for each patient sample andeach QC sample.

Place each test tube in a rack and on top place its matching SPEcartridge.

To each SPE cartridge add 2.5 ml 90% Methanol with the repeaterdispenser. This elutes the cholate compounds which are collected intothe test tubes.

Place the test tubes in the Speed-Vac and centrifuge under vacuum withhigh heat for 45 min to reduce eluate volume and to remove methanolwhich interferes with ether extraction.

To each tube from the Speed-Vac and to each of the STD tubes, add 0.5 mlof 0.2 N HCl with the Eppendorf Repeater Pipette and vortex 30 sec. Thisacidification converts the cholate compounds into their free acid formfor ether extraction.

In the fume hood, to each tube add 3 ml of diethyl ether and vortexvigorously for 30 sec. This extracts the free acid form of the cholatecompounds into the ether phase.

Centrifuge 5 minutes at a minimum of 5000 rpm to accelerate phaseseparation.

Label another set of test tubes (13×100) one for each sample.

Carefully collect the upper ether layer and transfer to the new testtubes.

Place the ether extracts in the Speed-Vac vented to the fume hood andcentrifuge under vacuum without heat until samples are dry.Alternatively, samples can be dried with a gentle stream of N₂ gas.

Add 100 ul Mobile Phase to dried samples, vortex 30 sec and sonicate.

Transfer samples to Agilent 1.5 ml vials and cap.

HPLC/MS Parameters and System Preparation

Reagents, Supplies and Equipment

The following reagents are prepared and used in the HPLC-MS sampleanalysis.

Water, Clinical Laboratory Reagent Water (CLRW)

Methanol LCMS grade

10 mM Ammonium Acetate water

10 mM Ammonium Acetate methanol

Mobile Phase: 60% 10 mM Ammonium Acetate Methanol/40% 10 mM Ammonium

Acetate

Water

Volumetric flasks, appropriate sizes

Graduated cylinder

The following instruments and supplies are used in the HPLC-MS sampleanalysis.

Calibrated Analytical Balance

HPLC/MS instrument: Agilent 1100 series Liquid Chromatograph MassSpectrometer equipped with a G1956A multi-mode source, automaticsampler, HP Chemstation Software or equivalent.

Agilent Eclipse XDB C8, 2.1×100 mm 3.5 um liquid chromatograph column

Solvent Filter Degasser

0.22 μm nylon filters

The solvents and mobile phase are each prepared, filtered with a 0.22 μmnylon filter and degassed. Solvents and mobile phase each expire 48hours after preparation.

The LCMS system is prepared and tuned; the column is stabilized at 40°C. and conditioned by running the mobile phase for 30 min. The samplesare loaded to the autosampler. The column flow rate is 0.4 ml/min ofisocratic mobile phase buffer; 60% 10 mM Ammonium Acetate Methanol/40%10 mM Ammonium Acetate Water. 5 microliters of each sample is injectedby the autosampler. The MS is run in multimode electrospray (MM-ES)ionization with atmospheric pressure chemical ionization (APCI)ionization. Selected ion monitoring is performed at 407.30, 408.30 and411.30 m/z. Peaks are integrated by the system software. Three QCsamples are assayed with each analytical run. The concentration of theQC samples must fall within 15% accuracy.

Data from selective ion monitoring of either or both intravenous andoral samples are used to generate individualized oral and intravenousclearance curves for the patient. The curves are integrated along theirrespective valid time ranges and an area is generated for each.Comparison of intravenous and oral cholate clearance curves allowsdetermination of first-pass hepatic elimination or portal shunt. Theliver shunt fraction calculated by the formula:ShuntFraction=[AUC_(oral)/AUC_(IV)]*[Dose_(IV)/Dose_(oral)]*100%.wherein AUC represents area under the curve and Dose represents theamount (in mg) of dose administered.

Example 5. Slow, Moderate, and Rapid Progressors: Three DistinctCategories of Patients with Primary Sclerosing Cholangitis Detected byFunctional Assessment Using Cholate Testing

The current example examined the relationships of Portal HFR and SHUNTto patient age to estimate approximate rate of PSC disease progression.Primary Sclerosing Cholangitis (PSC) exhibits inexorable progression butthe rate of progression varies between patients. Cholate Testing canmeasure the Portal Hepatic Filtration Rate (HFR) and portal-systemicSHUNT, which correlate with varices, ascites, decompensation, and needfor transplant (Gastroenterology 2012, 142: 5911; Liver Transplantation2012, 18: S233). Surprisingly, the relationships of Portal HFR (FLOW)and SHUNT to patient age were found to be useful to estimate theapproximate rate of PSC disease progression. Cholate testing wasemployed to distinguish slow, moderate and rapid progressors.

Methods. PSC patients (n=42) and 32 healthy controls, ranging in agefrom 20 to 67, underwent Cholate Testing and medical histories wererecorded. Patients were subjected to two cholate testing methods.Specifically, a first distinguishable cholate was administered orallyand a second distinguishable cholate was administered intravenously. Theorally administered cholate, 2,2,4,4-d4 cholate (40 mg dose), was takenup directly into the portal vein by specific enteric transporters. Theintravenously administered cholate, 24-¹³C-cholate (20 mg), wasdistributed systemically and enters the liver primarily through thehepatic artery. Specific hepatic transporters clear the dual cholatesfrom the portal and systemic circulation. Peripheral blood samples werecollected at 0, 5, 20, 45, 60 and 90 minutes following administration.The distinguishable cholates in each blood sample were assayed by LCMSmethods. In this case the methods had been validated according to FDAguidelines for accuracy and precision. The values obtained from eachsample were used to determine portal hepatic flow rate (portal HFR,FLOW) and SHUNT by the methods of Everson, US 2010/0055734, Methods forDiagnosis and Intervention of Hepatic Disorders, filed Sep. 11, 2009;and Everson et al., US2008/0279766, Methods for Diagnosis andIntervention of Hepatic Disorders, filed Jan. 26, 2006; each of which isincorporated herein by reference. Oral cholate-2,2,4,4-d4 targets theportal circulation, and its clearance defines Portal HFR. IVcholate-24-¹³C clearance measures Systemic HFR. The ratio of Systemic toPortal HFR defines SHUNT. Reproducibility of duplicate testing wasexcellent with CVs of 10%; the averages of duplicate tests were used foranalysis.

Results. In PSC patients Portal HFR decreased and SHUNT increased withage, compared to healthy controls. Examination of these age-relatedchanges revealed 3 categories of PSC patients defined by drop in PortalHFR or increase in SHUNT—Slow, Moderate, and Rapid Progressors. Withineach category the age-related decline in function was approximatelylinear. Slow Progressors (n=23, 61%) demonstrated modest age-relateddecline in Portal HFR or increase in SHUNT. If they developed portalhypertension (PHTN), manifestations occurred later in life—the 3 withsplenomegaly were over age 45 and the 2 with varices were over age 62.Moderate Progressors (n=12, 32%) often developed portal hypertension (9out of 12 patients) and experienced decline earlier in life—splenomegalyas early as age 30 and varices as early as age 32. Most of the ModerateProgressors over age 54 had ascites and/or variceal bleeding (4 out of 5patients). Rapid Progressors (n=3, 8%) were relatively rare but thesepatients exhibited low Portal HFR and high SHUNT earlier than age 30.The patient A with the highest SHUNT in the category of ModerateProgressors received a liver transplant at age 58 while the patient Bwith the highest SHUNT in the category of Rapid Progressors received aliver transplant at age 20, as shown in FIGS. 2-4. FIG. 2 shows PSCpatients segregated into 3 distinct groups based on their Portal HFRtest values and age at the time of testing. FIG. 3 shows PSC patientssegregated into 3 distinct groups based SHUNT test values and age at thetime of testing. PSC Patients categorized as Slow Progressors had onlymodest changes in function compared to controls. PSC patientscategorized as Moderate and Rapid Progressors exhibited morecomplications and at earlier ages.

Each category of patients exhibited a unique pattern of cytokinessuggesting unique pathophysiological mechanisms, as shown in FIG. 5.IFN-γ did not change between controls and slow, moderate and rapidprogressors. TNF-α was increased in each category of PSC patientscompared to healthy controls. GM-CSF was increased in moderate and rapidprogressors compared to controls and slow progressors. IL-1b appeared totrend higher in rapid progressors, but was not statisticallysignificant. IL-6 was virtually undetected in healthy controls, whileslow, moderate and rapid progressors exhibited a trend to escalatinglevels of IL-6. IL-8 was virtually undetected in healthy controls, whileslow, moderate and rapid progressors exhibited a trend to escalatinglevels of IL-8.

Conclusions. Functional assessment by Cholate Testing identified 3distinct categories of patients with PSC: Slow, Moderate, and RapidProgressors. Differentiating categories of PSC patients by CholateTesting could enhance investigation of unique pathophysiologicmechanisms of disease progression and aid development of appropriatelytargeted therapy.

Example 6. An Algorithm Using SHUNT and Portal HFR to Categorize PSCPatients

PSC patients are assigned to a subcategory of PSC based on their SHUNTand Portal HFR and their age at the time of testing by the followingalgorithm.

Step 1 is based on SHUNT and age. If the patient's SHUNT (in percent)divided by their age (years) is greater than 1.7 then the patient isdiagnosed as a Rapid Progressor. If this value is less than 1.7, thenproceed to Step 2. FIG. 6 shows SHUNT vs Age Determines the RapidProgressors. The line starting at age 0 demarcates a SHUNT/age of 1.7and those above the line are Rapid Progressors and those below are otherPSC categories.

Step 2 is based on Portal HFR and age. If the Portal HFR (inmL/min/kg)+[0.35×age (years)] is greater than 29 then the patient isdiagnosed as a Slow Progressor. If this value is less than 29 then thepatient is diagnosed as a Moderate Progressor. Those patients alreadydiagnosed as a Rapid Progressor in Step 1 are not further evaluated inthis manner by Portal HFR. FIG. 7 shows Portal HFR vs Age Determines theSlow and Moderate Progressors. The line starting at age 0 demarcates aPortal HFR+[0.35×age] that is equal to 29. Those patients above the lineare the Slow Progressors and those patients below this line are theModerate Progressors.

Example 7. A Method Using STAT to Categorize PSC Patients

The STAT test is a simplified screening test as described herein wherethe value is obtained from distinguishable cholate compound blood orserum concentration (in μM) obtained from a patient at a single timepoint (e.g., 60 minutes) after oral administration. The STAT test valuecan be used to estimate Portal blood flow. The STAT test value in PSCpatients can be compared to a cut-off value to categorize PSCprogression in most patients. Most PSC patients can be assigned to asubcategory of PSC based on simple STAT cutoffs and their age at thetime of testing, as shown in FIG. 8. FIG. 8 shows STAT can categorizemost PSC patients. Patients can be categorized using simple cutoffsshown as dark lines on this plot of Slow, Moderate, and RapidProgressors. Patients between 30 and 40 have overlapping STAT resultsand are not able to be categorized by this method. If patients are lessthan 30 and have STAT>1 μM the patient is diagnosed as a RapidProgressor. Patients in their 30s are not accurately categorized bySTAT. For older patients, cut-off values are shown in Table 8.

TABLE 8 STAT test cut-off values for PSC Patients. PSC Patient Age STATcut-off (μM) PSC Category <30 yrs >1 Rapid Progressor 30's N.A.40's >0.8 Moderate Progressor <0.8 Slow Progressor 50's >1.4 ModerateProgressor <1.4 Slow Progressor 60's >2.0 Moderate Progressor <2.0 SlowProgressor

Example 8. Development of a Disease Severity Index

Previously, the Portal HFR test or SHUNT test were each utilizedseparately in various cohorts of patients and normal controls to developcut-offs for specific chronic liver diseases or conditions. As describedherein, a disease severity index (DSI) is obtained from a DSI equationwhere the terms of the equation comprise one or more liver function testresult values selected from SHUNT, Portal HFR, and/or Systemic HFR,where test result values are used as terms in a disease severity indexequation to calculate a Disease Severity Index (DSI).

In one embodiment, the utility of the DSI in predicted response(sustained virologic response, SVR) of a group of CHC patients topegylated interferon/ribavirin (PEG/RBV) treatment was investigated. The% of CHC patients that achieved SVR was calculated for groups ofpatients within specified ranges of DSI, as shown in Tables 9-11. A DSIequation was developed incorporating both the SHUNT and portal HFR testvalues. The DSI equation is shown below.DSI=9.84 (SHUNT)−12.36 LOGe (portal HFR)+50.5

Applying the DSI equation to a group of healthy controls gave a meanscore+/−SD of 10+/−3. The most functionally impaired patient, whorequired a transplant within 3 weeks of testing, had a score of 47. Thehighest score in a patient that might survive is expected to be about50. Use of DSI compared to SHUNT or Portal HFR alone is shown in Tables9-11, which show the % of CHC patients that achieved SVR calculated forgroups of patients within specified ranges of SHUNT, Portal HFR, or DSI.

TABLE 9 SHUNT and SVR in CHC Patients. SHUNT <25 25-35 35-45 45-60 >60all % SVR 17% 14% 14% 3% 5% 14% SVR 11 12  7  1  1  32 N 63 59 50 37 21230

TABLE 10 Portal HFR and SVR in CHC Patients. HFR >20 15-20 10-15 5-10 <5all % SVR 15% 21% 17% 7% 0% 14% SVR  5 11 12  4  0  32 N 34 52 70 59 15230

TABLE 11 DSI and SVR in CHC Patients. DSI 0-20 20-25 25-30 30-40 40-50all % SVR 19% 16% 15% 0% 0% 14% SVR 17 10  5  0 0  32 N 89 62 33 37 9230

In some embodiments, the DSI is used to predict response to an antiviraltreatment, e.g., % of patients with CHC who will achieve SVR. In someembodiments, the DSI is used to predict the response to treatment, e.g.,% of patients with CHC who will achieve SVR following treatment withPEG/RBV.

Example 9. Disease Severity Index to Assess Liver Related Outcomes

Nonalcoholic fatty liver disease (NAFLD) encompasses benign hepaticsteatosis (fatty liver) and steatosis accompanied by inflammation,necrosis, and fibrosis (NASH) which may progress to cirrhosis andclinical complications. Although NASH is an aggressive necroinflammatoryprocess, rates of progression of fibrosis and evolution to cirrhosisvary greatly between patient.

The slow rate of progression dictates that many years of observation andfollowup would be required to establish the natural history of NASH orto prove that a treatment or intervention reduces clinical outcomes.Long-term studies with the primary outcome of clinical complications areenormously costly and suffer from both patient and investigatorattrition. Short-term studies using early markers or surrogates thatcorrelate with clinical outcomes are desirable.

Until now, fibrosis stage on liver biopsy was considered the goldstandard as the surrogate for clinical outcomes. Several studies havedemonstrated that severity of fibrosis, but not steatosis, predictsfuture risk for clinical outcome.

An alternative to liver biopsy should be accurate, reproducible,well-tolerated, relatively inexpensive, and noninvasive. In addition,these alternative tests should correlate with fibrosis, independent ofthe degree of steatosis, and predict risk for clinical outcome.Quantitative liver function tests (QLFTs) were developed to addressthese needs. Nearly all QLFTs satisfy one or more criteria asalternatives to liver biopsy. Dual cholate clearance (SHUNT) satisfiesall of these criteria. The NIH- and industry (Roche)-sponsored QLFTancillary study of the Hepatitis C Antiviral Long-Term Treatment AgainstCirrhosis (HALT-C) Trial evaluated the ability of QLFTs to predictclinical outcomes.

Patients (N=285) were studied with a battery of QLFTs (caffeine,antipyrine, lidocaine-MEGX, galactose, dual cholates, and SPECTliver-spleen scan) at baseline, 2 yr, and 4 yr, and followed forclinical outcomes (Child-Turcotte-Pugh (CTP) increase, varices,encephalopathy, ascites, liver related death) for up to 8.3 years(4.9±2.2 yrs, mean±SD). The likelihood of clinical outcome for thosebeyond the high-risk cutoff for each test was compared. Hazard ratiosfor baseline tests ranged from 6.5 for oral cholate to 2.2 for GEC, andpooled relative risk for serial tests ranged from 14.1 for oral cholateto 2.5 for MEGX. In additional publications and presentations, the dualcholate test was shown to not only predict clinical outcome, but alsocorrelated with stage of fibrosis, risk for cirrhosis, risk for varices,and variceal size and tracked improvement in liver function after SVRand recovery from living liver donation.

Interestingly, in models of dual cholate predicting clinical outcome,histologic stage of fibrosis dropped from significance; thereforestaging liver biopsy could surprisingly be avoided. Given the broadclinical associations, further investigation of the potential utility ofdual cholate testing as an alternative to biopsy seemed warranted.

The initial development of dual cholate into a clinical test requiredsimplification of clinical testing, revision of laboratory procedures,institution of quality controls, and validation of testing performancecharacteristics according to FDA guidelines for bioanalytical proceduresand method.

A Data-Use Agreement with NIH and HALT-C was reached to re-examine andexplore clinical relationships of the revised test. Three majorparameters of liver function are defined by the test: Systemic HepaticFiltration Rate (HFR) from clearance of intravenously administeredcholate, Portal HFR from orally administered cholate, and SHUNT from theratio of clearances. The disease severity index is a composite of allthree variables. It was defined from the ability of these tests topredict risk for future clinical outcome in patients with fibroticstages of chronic hepatitis C, as shown in Example 10.

Using clinical outcome as the endpoint in HCV patients a diseaseseverity index (DSI) was defined from the parameters of the test, asshown in Example 10. The DSI correlates with liver disease severity inHCV patients, PSC patients, and in NASH patients in a new studycurrently enrolling subjects. DSI is highly reproducible, correlateswith fibrosis, and predicts clinical outcome. FIG. 10 demonstrates thatDSI linearly correlates with fibrosis (left panel) but is not influencedby steatosis (biopsy fat score, right panel). The performance of DSI inidentifying the patients with future clinical outcomes was compared tothat of Ishak fibrosis score, platelet count, and MELD, (FIG. 11 andTable 12).

TABLE 12 Identifying Patients who will have Outcomes. Test ParameterC-statistic Optimum Cut-off Cholate DSI 0.83 >23 Biopsy Ishak Fibrosis0.75 >F4 CBC Platelets 0.75 <140 Std Labs MELD 0.70 >6

As shown in FIG. 11 and Table 12, at the optimum cutoffs DSI had thehighest sensitivity, specificity, PPV, and NPV when compared to biopsy(Ishak fibrosis), CBC (platelets), or standard labs (MELD). QLFTs,particularly dual cholate, can be an accurate, reproducible,cost-effective, noninvasive alternative to liver biopsy as an endpointin studies of natural history or for monitoring the effectiveness oftreatment in NAFLD.

Example 10. Disease Severity Index Assessment of Liver Related Outcomesin Chronic Hepatitis C

Cholate testing was performed at baseline in 224 chronic HCV patients(Ishak F2-F6) enrolled in the HALT-C trial, characterized by CTP scoresof 5 or 6 and no prior history of clinical complications.

Specifically, archive serum was re-analyzed to determine cholateclearance curves for systemic Hepatic Filtration Rate (HFR) fromclearance of intravenously administered cholate, Portal HFR from orallyadministered cholate, and SHUNT from the ratio of clearances using animproved LCMS method validated to FDA guidelines. Patients were followedfor clinical outcomes for up to 8.3 years (4.9±2.2 years, mean±SD).Clinical outcomes (n=54) were defined as CTP progression, varicealhemorrhage, ascites, hepatic encephalopathy, or liver-related death.

Derivation of a Disease Severity Index (DSI) was performed usingunivariate Cox univariate Cox proportional hazard regression analysis asshown in Table 13.

TABLE 13 Univariate Cox Proportional Analysis Hazard RegressionAnalysis. Chi-Square Chi-Square SHUNT 44.7 Log_(e) SHUNT 34.8 Portal HFR51.2 Log_(e) Portal HFR 70.2 Systemic HFR 21.9 Log_(e) Systemic HFR 29.8

The tests with the highest Chi-square were combined into a diseaseseverity index to improve performance.DSI=A(SHUNT)+B(log_(e) portal HFR)+C(log_(e) systemic HFR)+D.

A DSI equation was developed based on prediction of first clinicaloutcome in the HALT-C cohort:DSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50

ROC curves for predicting outcomes for SHUNT, Portal HFR and DSI wereprepared and are shown in FIG. 12. Optimum cut-offs were determined asthe point on each curve closest to the top-left corner as shown in FIG.12.

Patients ranged from DSI 9 (normal) to 40 (severe dysfunction). ROCcurves showed that DSI could identify patients with medium/largevarices, c-statistic 0.82, and could predict which patients would haveclinical outcomes, c-statistic 0.83, and DSI>25 was the optimum cutofffor both. DSI>25 had a higher balanced accuracy than cirrhosis by biopsy(Ishak F5-F6) and the PPV for identifying medium/large varices increased41% relative to biopsy and the PPV for predicting outcomes increased 47%(Table 14).

TABLE 14 Prognostic Test Performance. Balanced Test Cutoff SensitivitySpecificity PPV NPV Accuracy Biopsy (Ishak F5-F6) 72% 66% 40% 88% 69%SHUNT > 44% 59% 81% 50% 86% 70% Portal HFR < 9.7 74% 81% 55% 91% 77%mL/min/kg DSI > 25 74% 84% 60% 91% 79%

The prognostic test performance of each test system was evaluated andthe highest sensitivity, specificity, PPV, NPV, and balanced accuracywere all achieved by the cholate test based DSI as shown in Table 14.

FIG. 13 shows a plot for non-cirrhotic patients (Ishak F2,3,4; n=19, 63,45) with mild disease, moderate disease and severe disease test resultsof cholate based tests SHUNT (%), systemic HFR (mL/min/kg), portal HFR(mL/min/kg) and DSI according to this example. Black circles indicatepatients with clinical outcomes, light grey circles indicate patientswithout clinical outcomes; and dark grey circles indicate normal healthycontrols. Surprisingly, non-cirrhotic patients (Ishak F2, 3, 4) withhigh DSI have greater risk of outcomes.

FIG. 14 shows a plot for cirrhotic patients (Ishak F5, 6; n=48,49) withmild disease, moderate disease and severe disease test results ofcholate based tests SHUNT (%), systemic HFR (mL/min/kg), portal HFR(mL/min/kg) and DSI according to this example. Black circles indicatepatients with clinical outcomes, light grey circles indicate patientswithout clinical outcomes; and dark grey circles indicate normal healthycontrols. Surprisingly, cirrhotic patients (Ishak F5,6) with low DSIhave a lower risk of clinical outcomes.

Remarkably, cholate tests could outperform biopsy diagnosed cirrhosis inpredicting clinical outcomes in hepatitis C patients. The highestprognostic performance was achieved by combining cholate test resultsinto a DSI. This example shows that dual cholate liver function testyielding a DSI could outperform histologic fibrosis stage in identifyingpatients with medium/large varices and in predicting clinical outcomesin chronic HCV patients.

Example 11. Disease Severity Index Assessment of Disease Severity inPrimary Sclerosing Cholangitis (PSC)

Cholate testing was compared to MELD in the assessment of diseaseseverity in a group of compensated or minimally decompensated patientswith Primary Sclerosing Cholangitis (PSC).

20 healthy controls served as a reference for comparing the cholate testresults in the PSC population. 43 PSC patients were enrolled that hadbeen diagnosed by cholangiography Most were of middle age, male, and hadlow MELD, CTP, or Mayo PSC Risk Scores. The mean values for bilirubin,INR, albumin, and platelet count reflect the relatively compensatedstage of their disease. Alkaline phosphatase was modestly elevated witha broad range. Specifically, the 43 PSC patients were avg. 48.4±12.9 yrsof age, 76% male, with avg. MELD scores of 9.5±4.3, avg. CTP scores of6.0±1.5, avg. Mayo PSC risk score of 0.85±0.74, avg. bilirubin 1.8±1.8mg/dL, avg. INR 1.2±0.6, avg. albumin 3.6±0.5, avg. platelet count190±95×10⁻³ uL-1, and avg. alkaline phosphatase of 206±196 IU/mL.

Patients were subjected to overnight fast and each patient was studiedtwice within two weeks to check reproducibility. Chlorate tested wasperformed by simultaneous dual administration of 20 mg [24-¹³C]-cholatemixed with HSA intravenously, and 40 mg [2,2,4,4-²H]-cholate in juiceorally. Blood samples were taken at 0, 5, 20, 45, 60, and 90 minfollowing cholate administration. The serum samples were analyzed byHPLC/MS. Clearances (IV and PO), SHUNT, Disease Severity Index (DSI)were calculated from serum cholates. The cholate oral clearance and thecholate clearance after intravenous administration were calculated fromthe dose (40 mg for oral administration and 20 mg for intravenousadministration) divided by the area under the concentration-time curvesfor each isotope (milligrams per minute per milliliter) and normalizedfor the body weight (kilograms), and the cholate shunt was the ratio ofclearances for intravenously and orally administered isotopes. The DSIwas calculated from the DSI equation developed based on prediction offirst clinical outcome in the HALT-C cohort, as shown in Example 10.DSI=5.75 (SHUNT)−7.22 (Log_(e) Portal HFR)−8.45 (Log_(e) SystemicHFR)+50.

Cholate testing demonstrated excellent reproducibility with very lowvariably from one testing date to another. Reproducibility data areshown in Table 15.

TABLE 15 Reproducibility of Cholate Testing SHUNT Portal HFR SystemicHFR Correlation Coefficient R² 0.96 0.95 0.90 Coefficient of VariationCV 10.1% 10.7% 9.7% Intraclass Correlation ICC 0.93 0.90 0.80Coefficient

The correlation coefficients were excellent at 0.95 and 0.97. Theaverage CV for HFR and SHUNT are 10%, and for STAT was 21%. As STAT is aone-time measurement rather than average of several points, we expecteda higher CV. Importantly, there was no significant change in CV acrossthe range of test results indicating excellent reproducibility acrosswide range of disease severity. The data show that cholate testing isreproducible.

The intra-class correlation measures variability of an individual overthe range of all test results. The ICCs range between 0.9 to 0.94,indicating within individual variability is very low. To put this intocontext, ICC of 0.7-0.8 indicates strong agreement between tests.

DSI identified PSC patients with varices or decompensation.

FIG. 15 shows a plot of cholate test results for DSC patients andhealthy controls. Portal HFR is plotted on the X axis and systemic HFRon the Y axis. SHUNT, the ratio of systemic to portal HFR is representedby the diagonal lines. DSI is displayed in shaded regions—light greyzone in the upper right with a cutoff of DSI=14 shows DSI of healthycontrols, the white zone with a cutoff of DSI=18 shows the DSI of milddisease, the medium grey zone with the cutoff of DSI=36 shows DSI ofmoderate disease, and the dark grey zone at the lower left shows DSI forthe most severe disease. Predictive Cutoffs for PSC disease, varices,and decompensation are shown at the interfaces between zones.

Performance of the cholate cutoffs for defining patients at-risk forvarices or decompensation are shown in Table 16.

TABLE 16 Performance of Cholate Cutoffs from PSC Patients. Balanced PSCPatients with cutoff Sens. Spec. PPV NPV Accuracy Varices (n = 13) DSI≥18 100% 70% 59% 100% 85% Decompensation DSI ≥36 100% 100% 100% 100%100% (n = 4)

In healthy controls, DSI was 10±3. In PSC, DSI could identify patientswith PHTN (ROC c-statistic 0.80) and varices (ROC c-statistic 0.93). ADSI≥18 was optimal (balanced accuracy 82%) for identifying PHTN andcould also identify all patients with varices (100% sensitivity, 70%specificity, 59% PPV, 100% NPV). This cutoff marked the boundary betweenmild and moderate disease. All patients with DSI≥36, separating moderateand severe disease, either had medium varices or had suffered a varicealhemorrhage, and 75% had ascites. No patients with DSI <36 had ascites orvariceal hemorrhage.

DSI quantifies PSC disease severity. As shown in FIG. 16, Panel A showsa comparison of DSI with MELD for all the PSC patients. The DSI ofhealthy controls is also shown for reference. DSI separates PSC patientsfrom controls, PSC patients without clinical manifestations from healthycontrols and PSC patients with varices. DSI identifies the sickestpatents, who developed decompensation. In contrast, there wassignificant overlap in MELD scores between these groups of PSC patients.MELD could not distinguish the groups with any degree of certainty.Surprisingly, cholate testing DSI is superior to MELD in assessingdisease severity in patients with primary sclerosing cholangitis.

FIG. 16 Panels B and C show DSI compared to MELD only in the listedpatients-patients on the waiting list for liver transplant.

FIG. 16 Panel B shows a DSI of approximately 20 clearly separates thePSC patients with varices from the PSC patients without varices.

FIG. 16 Panel C shows a DSI of approximately 35 clearly separates thePSC patients with decompensation from those without decompensation.

In contrast, FIG. 16 Panels B and C show the overlap in MELD scoresbetween these groups of PSC patients indicate MELD could not distinguishthe patients with varices or the patients with decomp with any degree ofcertainty.

This example shows DSI is superior to MELD in assessing PSC diseaseseverity and identifying patients at risk for varices ordecompensation-especially in patients with lower MELD scores. DSI couldbe used to adjust priority for liver transplantation for PSC patients onthe Waiting List.

FIG. 17 shows changes in DSI and disease severity in PSC patients thatwere assessed after a 1 year follow-up. The change in DSI was plottedagainst the age of the patient as shown in FIG. 17. Serial DSImeasurements defined category of disease severity. Patients slow PSCprogression are shown in the left panel where a cutoff=18 is indicativeof PHTN. Patients with moderate and rapid PSC progression are shown inthe right panel where a DSI cutoff=35 is indicative of decompensation.

Example 12. Cholate Testing and Disease Severity Index Identification ofPrimary Sclerosing Cholangitis Waiting List Patients at Risk forClinical Complications

MELD may not be able to assess the risks for clinical complications inlisted PSC patients compared to a disease severity index (DSI) based ondual cholate clearances and shunt. Cholate testing was compared to MELDin the assessment of disease severity in a group of patients withPrimary Sclerosing Cholangitis (PSC) on the waiting list for livertransplantation.

Patients were tested as provided in Example 11. DSI was calculated asprovided in Example 11. Of the 43 PSC patients tested, 10 were on thewaiting list for LT. The PSC patients were compared to 20 healthycontrols. A comparison of cholate test values, DSI and MELD for patientsand healthy controls is shown in Table 17.

TABLE 17 Cholate and DSI values compared to MELD scores in PSC Patientsand Healthy Controls. SHUNT Portal HFR MELD n (%) (mL/min/kg) DSI ScoreHealthy 20 20 ± 1 34.7 ± 1.7  9.7 ± 0.7 Controls PSC Patients 43 43 ± 314.2 ± 1.0 21.1 ± 1.2 8.7 ± 0.5 P < 0.001  P < 0.001 P < 0.001 PSC notlisted 33 40 ± 3 15.4 ± 1.1 19.8 ± 1.3 7.8 ± 0.3 for LT Listed PSC 10 56± 7 10.2 ± 1.8 25.5 ± 3.1 11.4 ± 1.4  P < 0.01  P < 0.05 P < 0.05  P <0.001 Listed PSC w/o 5 41 ± 6 15.0 ± 1.3 18.0 ± 1.1 9.0 ± 1.1 varicesListed PSC w 5 71 ± 8  5.4 ± 1.4 33.0 ± 3.7 13.8 ± 2.1  varices P <0.005 P < 0.05 P < 0.005 ns

Table 17 shows SHUNT, Portal HFR and DSI couls differentiate PSCpatients from healthy controls, listed PSC patients from PSC patientsnot listed for liver transplant (LT), listed PSC patients with varicesfrom those without varices. MELD could not differentiate those withvarices.

FIG. 18 shows a plot of DSI versus MELD scores in PSC patients on thewaiting list for liver transplantation. DSI was superior to MELD inassessing risk for complications and priority for liver transplant inPSC patients. Despite low MELD scores, PSC patients with DSI>20developed portal hypertension-related complications, and PSC patientswith DSI>40 required liver transplantation.

Example 13. Cholate Testing and Disease Severity Index Measurement ofFunctional Improvement after Sustained Virological Response in ChronicHepatitis C Patients

Chronic HCV patients with advanced fibrosis or cirrhosis are difficultto treat and cure. The aims of this study were to determine if liverfunction measured with Cholate Testing could predict sustainedvirological response (SVR) to peginterferon/ribavirin (PEG/RBV) and tomeasure the improvement in hepatic function in those achieving SVR.

230 chronic HCV patients (Ishak F2-6) enrolled in the HALT-C Trial,characterized by advanced fibrosis and failure of prior treatment withinterferon-based treatment, were tested at baseline and then retreatedwith PEG/RBV. Patients achieving sustained virological response SVR(n=32, including 5 cirrhotics) and non-responders (NR) were retested at2 yrs.

At baseline and after 2 years, patients were subjected to chloratetesting by simultaneous dual administration of 20 mg [24¹³C]-cholatemixed with HSA intravenously, and 40 mg [2,2,4,4-²H]-cholate in juiceorally. Blood samples were taken at 0, 5, 20, 45, 60, and 90 minfollowing cholate administration. The serum samples were analyzed byHPLC/MS, and clearances (IV and PO), SHUNT, Disease Severity Index (DSI)were calculated from serum cholates. The DSI was calculated from the DSIequation developed based on prediction of first clinical outcome in theHALT-C cohort, as shown in Example 10.

FIG. 19-panel-A shows a graph of patients achieving SVR compared toquartiles for hepatic function. The probability of SVR correlated bestwith DSI.

As shown in Table 18, hepatic functional declined in non-responders (NR,n=80), and improved with SVR (n=23).

TABLE 18 Hepatic Function Change from Baseline in HCV Patients with andwithout SVR following Retreatment. Change (mean ± SEM) NR SVR P SHUNT(%)  5.5 ± 1.9 −6.1 ± 1.9 P < 0.01 Portal HFR −1.3 ± 0.6  4.1 ± 1.0 P <0.0001 (mL/min/kg) Systemic HFR −0.1 ± 0.1  0.0 ± 0.2 ns (mL/min · kg)DSI  1.8 ± 0.6 −2.4 ± 0.6 P < 0.001

FIG. 19B shows hepatic functional improvement after SVR followingretreatment of chronic HCV patients with PEG/RBV(peginterferon/ribavirin). More severe baseline impairment resulted ingreater functional improvement after SVR when tested two years afterbaseline.

Non-invasive cholate liver function testing predicts the probability ofachieving SVR when patients are retreated with PEG/RBV and quantifiessignificant improvements in function after SVR. The improvements inhepatic function after SVR are greater in those patients who had moresevere baseline impairment.

Example 14. Development of a Disease Severity Index Equation

The aim of this study was to compare dual cholate liver function testingto histologic stage of fibrosis in identifying those chronic HCVpatients who have medium/large varices and those who are at risk forfuture clinical outcomes.

Chronic HCV patients (n=220) enrolled in the HALTC trial had dualcholate testing, liver biopsy, endoscopic screening for varices, andwere followed for 4.9±2.2 years for clinical outcomes. The patients hadIshak fibrosis scores of F2-6, CTP scores of 5 or 6, and no priorhistory of clinical complications. Medium or large esophageal variceswere found in 22 patients. Clinical outcomes, defined as a 2 point CTPprogression, variceal bleeding, ascites, hepatic encephalopathy, orliver related death, occurred in 52 patients.

Dual cholate testing was performed. Briefly, orally administeredCholate-2,2,4,4-d4 (40 mg) is taken up into the portal vein by specificileal bile salt transporters. Intravenously administered Cholate-24-13C(20 mg) enters the liver primarily through the hepatic artery. Specifichepatic bile salt transporters clear the dual cholates from the portaland systemic circulation. Peripheral blood samples were taken from thepatients at 0, 5, 20, 45, 60, and 90 minutes after simultaneous dosingand were assayed by an LCMS method validated to FDA guidelines foraccuracy and precision. SHUNT, portal HFR and systemic HFR werecalculated.

The AUC c-statistics and optimum cutoffs were determined from ROCcurves, and test performance was evaluated by the balanced accuracy andYouden index (J). Performance of individual tests is shown in Table 19.Individual tests predict outcomes.

TABLE 19 Performance of Individualized Cholate Tests Youden AUC C-Optimum Balanced Index statistic cutoff Sens. Spec. PPV NPV Accuracy (J)Portal HFR 0.83 8.5 mL/min/kg 65% 88% 62% 89% 76% 0.53 SHUNT 0.75 46%58% 82% 50% 86% 70% 0.40 Systemic 0.73 3.6 mL/min/kg 60% 80% 48% 86% 70%0.39 HFR

Each test and its Log transform was also evaluated by Hazard Regressionas shown in Table 20.

TABLE 20 Univariate Cox Proportional Hazard Regression Analysis(Chi-square). Portal HFR 51 Log_(e) Portal HFR 70 SHUNT 45 Log_(e) SHUNT35 Systemic HFR 22 Log_(e) Systemic HFR 30

The tests with the highest Chi-square were combined into a diseaseseverity index to improve performance.

The test with highest Chi-square, Log_(e) Portal HFR, was graphedagainst the second best, SHUNT, on a new feature space. The optimumlinear classifier was:Y _((SHUNT))=1.24 X _((Loge Portal HFR))−2.36which defined the component weighting and the DSI₂.

DSI₂=1.24 Log_(e) Portal HFR−SHUNT

TABLE 21 DSI2 Statistics. Opti- Youden AUC C- mum Balanced Indexstatistic cutoff Sens. Spec. PPV NPV Accuracy (J) DSI2 0.82 2.36 73% 83%57% 91% 78% 0.56

DSI₂ had improved performance over the individual tests.

The DSI₂ was graphed against the Log_(e) Systemic HFR on a new featurespace. The optimum linear classifier was:Y _((Loge Systemic HFR))=−0.623 X _((DSI2))+3.03defining the weighting of the 3^(rd) component and the DSI₃.

DSI₃=Log_(e) Systemic HFR+0.623 DSI₂

TABLE 22 DSI3 Statistics. Opti- Youden AUC C- mum Balanced Indexstatistic cutoff Sens. Spec. PPV NPV Accuracy (J) DSI3 0.83 3.03 87% 72%49% 95% 79% 0.59

DSI₃ had improved performance over DSI₂.

The DSI₃ was rearranged and constants added to have the best liverfunction anticipated as DSI=0, and the worst liver function as DSI=50.DSI=5.34 SHUNT−6.65 Log_(e) Portal HFR−8.57 Log_(e) Systemic HFR+44.66

The DSI equation identifies patients with medium/large varices.Specifically, using the DSI equation, a DSI>19 indicates high risk ofmedium to large varices; DSI 10-19 is indicative of low risk ofmedium/large varices; and DSI of 0-10 is indicative of healthy liverfunction

The DSI equation predicts future clinical outcomes in patients.Specifically, using the DSI equation, a DSI>19 indicates high risk ofclinical outcomes; DSI 10-19 is indicative of low risk of clinicaloutcomes; and DSI of 0-10 is indicative of healthy liver function.

A comparison of DSI to fibrosis stage on biopsy is shown in Tables 23and 24.

TABLE 23 Identifying Patients with Medium/Large Varices. Youden AUC C-Optimum Balanced Index statistic cutoff Sens. Spec. PPV NPV Accuracy (J)Biopsy 0.79 Cirrhosis 77% 61% 18% 96% 69% 0.38 Fibrosis stage (IshakF5-F6) Dual Cholate 0.82 DSI >19 86% 65% 21% 98% 76% 0.51 DSIImprovement 3% 12% 6% 18% 2% 9% 33% over Biopsy

TABLE 24 Identifying Future Clinical Outcomes. Youden AUC C- OptimumBalanced Index statistic cutoff Sens. Spec. PPV NPV Accuracy (J) Biopsy0.75 Cirrhosis 71% 66% 39% 88% 69% 0.37 Fibrosis stage (Ishak F5-F6)Dual Cholate 0.83 DSI >19 87% 72% 49% 95% 79% 0.59 DSI Improvement 11%22% 9% 24% 7% 16% 57% over Biopsy

As shown in Tables 23 and 24, DSI is better than histologic fibrosisstage at biopsy in all measures of performance for identifying futureclinical outcomes.

We claim:
 1. A method of displaying likelihood or risk of futureclinical outcome, disease severity, and/or disease status in a patienthaving or at risk of a chronic liver disease, the method comprising a.obtaining distinguishable bile acid test results in the patientcomprising a portal hepatic filtration rate (portal HFR) value and asystemic hepatic filtration rate (systemic HFR) value from the patientat one or more test days; b. deriving one or more disease severity index(DSI) value(s) or mathematically transformed DSI value(s) of the patientfrom the portal and systemic HFR values or mathematically transformedportal and mathematically transformed systemic HFR values at the one ormore test days; c. plotting the DSI value(s) or mathematicallytransformed DSI value(s) of the patient on a two-dimensional graphicalplot, wherein the graphical plot is divided into a multiplicity ofdisease severity index (DSI) value zones predetermined by DSI values ormathematically transformed DSI values obtained from one or more groupsof known subjects having a known clinical outcome, disease severityand/or disease status; and d. determining the location of each DSI valueor mathematically transformed DSI value of the patient within the DSIvalue zones, wherein the location is indicative of likelihood or risk ofexperiencing a future clinical outcome, disease severity and/or diseasestatus in the patient, wherein the portal HFR test value in the patientis determined by a method comprising (i) receiving a plurality of bloodor serum samples collected from the patient having or at risk of achronic liver disease, following oral administration of a dose of adistinguishable bile acid (dose_(oral)) to the patient, wherein thesamples have been collected from the patient over intervals of a periodof time of no more than 180 minutes after administration; (ii) measuringconcentration of the distinguishable bile acid in each sample; (iii)generating an individualized oral clearance curve from the concentrationof the distinguishable bile acid in each sample comprising using acomputer algorithm curve fitting to a model distinguishable bile acidclearance curve; (iv) computing the area under the individualized oralclearance curve (AUC)(mg/mL/min) and dividing the dose (in mg) by AUC ofthe orally administered distinguishable bile acid to obtain the oraldistinguishable bile acid clearance in the patient; and (v) dividing theoral distinguishable bile acid clearance by the weight of the patient inkg to obtain the portal HFR value in the patient (mL/min/kg).
 2. Themethod of claim 1, wherein the one or more portal and systemic HFRvalues or mathematically transformed portal and systemic HFR values areobtained on the same test day.
 3. The method of claim 1, wherein theclinical outcome is selected from the group consisting ofChild-Turcotte-Pugh (CTP) progression, variceal hemorrhage, ascites,splenomegaly, varices, portal hypertension (PHTN), hepaticencephalopathy, decompensation, and liver-related death.
 4. The methodof claim 1, wherein the chronic liver disease is selected from the groupconsisting of chronic hepatitis C (CHC), chronic hepatitis B, alcoholicliver disease, Alcoholic SteatoHepatitis (ASH), Non-Alcoholic FattyLiver Disease (NAFLD), steatosis, Non-Alcoholic SteatoHepatitis (NASH),autoimmune liver disease, cryptogenic cirrhosis, hemochromatosis,Wilson's disease, alpha-1-antitrypsin deficiency, primary sclerosingcholangitis (PSC), cholestatic liver disease, and hepatocellularcarcinoma (HCC).
 5. The method of claim 4, wherein the chronic liverdisease is selected from the group consisting of CHC, PSC, and NASH. 6.The method of claim 1, wherein the DSI value zones on the plot definecutoffs delineating one or more of the group consisting of healthy liverfunction, mild disease, moderate disease, and severe disease.
 7. Themethod of claim 1, wherein changes in DSI values within the patient overtime are plotted on the graph and used to inform the patient of statusof likelihood or risk for future clinical outcomes, disease severity,and/or disease status.
 8. The method of claim 1, wherein the diseasestatus is selected from the group consisting of disease progression,disease improvement, need for treatment, effectiveness of treatment,disease prognosis, sustained virological response, instituting treatmentor intervention, endpoint in clinical trial, selection of a patient fortreatment, therapeutic intervention, or clinical trial., risk of hepaticdecompensation in patients with hepatocellular carcinoma (HCC) beingevaluated for hepatic resection, measuring functional impairment incholestatic liver disease., and prioritizing liver transplant in thepatient.
 9. The method of claim 8, wherein the treatment of chronicliver disease in the patient is selected from the group consisting ofantiviral treatment, antifibrotic treatment, antibiotics,immunosuppressive treatments, anti-cancer treatments, ursodeoxycholicacid, obesity treatment, diabetes mellitus treatment, insulin, insulinsensitizing agents, interventional treatment, liver transplant,lifestyle changes, dietary restrictions, low glycemic index diet,antioxidants, vitamin supplements, transjugular intrahepaticportosystemic shunt (TIPS), hepatic resection, catheter-directedthrombolysis, balloon dilation and stent placement, balloon-dilation anddrainage, weight loss, exercise, and avoidance of alcohol.
 10. Themethod of claim 1, wherein the DSI value zones are predetermined bycalculating a disease severity index (DSI) value in each subject in agroup of known subjects having a known clinical outcome, diseaseseverity and/or disease status, by a method comprising (a) obtaining oneor more liver function test values in each known subject in the group,wherein the one or more liver function test values are obtained from oneor more liver function tests selected from the group consisting ofcholate SHUNT test, portal hepatic filtration rate (portal HFR), andsystemic hepatic filtration rate (systemic HFR); (b) employing a diseaseseverity index equation (DSI equation) to obtain a DSI value for eachknown subject in the group, wherein the DSI equation comprises one ormore terms and a constant to obtain the DSI value, wherein at least oneterm of the DSI equation independently represents a liver function testvalue in the known subject from step (a) or a mathematically transformedliver function test value in the known subject from step (a); and the atleast one term of the DSI equation is multiplied by a coefficientspecific to the liver function test; and (c) performing statisticalcomparison of the DSI values from each known subject in the group toprovide one or more DSI cut-off values, and plotting the DSI cut-offvalues on the graphical plot to form the DSI value zone for the group ofknown subjects having a known clinical outcome, disease severity and/ordisease status.
 11. The method of claim 10, wherein the group of knownsubjects is selected from a group of healthy subjects, or a group ofsubjects with chronic liver disease having Ishak fibrosis score (liverbiopsy) F2 (mild portal fibrosis), F3, F4 (moderate bridging fibrosis),F5 (nodular formation and incomplete cirrhosis), or F6 (cirrhosis);portal hypertension; Childs-Turcotte-Pugh (CTP) score A; CTP score B,CTP score C; primary sclerosing cholangitis (PSC) not listed fortransplant; PSC listed for liver transplant; PSC listed for livertransplant without varices; PSC listed for liver transplant withvarices; ascites; stomal bleeding; splemomegaly; varices; varicealhemorrhage; hepatic encephalopathy, decompensation; or liver relateddeath.
 12. The method of claim 1, the samples have been collected fromthe patient at from two to seven time points over intervals of theperiod of time of no more than 180 minutes after administration.
 13. Themethod of claim 1, wherein the systemic HFR value in the patient isdetermined by a method comprising (a) receiving a plurality of blood orserum samples collected from a patient having or at risk of a chronicliver disease, following intravenous administration of a dose of adistinguishable bile acid (dose_(iv);) to the patient, wherein thesamples have been collected from the patient over intervals spanning aperiod of time of no more than 180 minutes after administration; (b)measuring concentration of the distinguishable bile acid in each sample;(c) generating an individualized intravenous clearance curve from theconcentration of the distinguishable cholate in each sample comprisingusing a computer algorithm curve fitting to a model distinguishable bileacid clearance curve; (d) computing the area under the individualizedintravenous clearance curve (AUC)(mg/mL/min) and dividing the dose (inmg) by AUC of the intravenously administered distinguishable bile acidto obtain the intravenous distinguishable bile acid clearance in thepatient; and (e) dividing the intravenous distinguishable bile acidclearance by the weight of the patient in kg to obtain the systemic HFRvalue in the patient (mL/min/kg).
 14. The method of claim 1, wherein thederiving DSI value comprises employing a disease severity index equation(DSI equation) to obtain a DSI value or mathematically transformed DSIvalue in the patient, wherein the DSI equation comprises one or moreportal HFR or mathematically transformed portal HFR terms, one or moresystemic HFR or mathematically derived systemic HFR terms, and aconstant to obtain the DSI value.
 15. The method of claim 14, whereinthe disease severity index (DSI) equation comprisesDSI=A(SHUNT)+B(log_(e) portal HFR)+C(log_(e) systemic HFR)+D whereinSHUNT is SHUNT test value in the patient (%); portal HFR is portalhepatic flow rate (HFR) test value in the patient as mL/min/kg, whereinkg is body weight of the patient; systemic HFR is systemic HFR value inthe patient as mL/min/kg, wherein kg is body weight of the patient; A isa SHUNT coefficient; B is a Portal HFR coefficient; C is a Systemic HFRcoefficient; and D is the constant.
 16. The method of claim 15, whereinthe SHUNT, the portal HFR, and/or the systemic HFR test values in thepatient were obtained on the same day.
 17. The method of claim 15,wherein the SHUNT coefficient is a number from 0to positive 25; thePortal HFR coefficient is a number from 0 to negative 25; and theSystemic HFR coefficient is a number from 0 to negative
 25. 18. Themethod of claim 15, wherein the constant is a positive number between 5and
 125. 19. The method of claim 1, wherein the distinguishable bileacid is a distinguishable cholate compound.
 20. The method of claim 19,wherein the distinguishable cholate compound is selected from the groupconsisting of a distinguishable cholic acid, a distinguishable glycineconjugate of cholic acid, a distinguishable taurine conjugate of cholicacid; a distinguishable chenodeoxycholic acid, a distinguishable glycineconjugate of chenodeoxycholic acid, a distinguishable taurine conjugateof chenodeoxycholic acid; a distinguishable deoxycholic acid, adistinguishable glycine conjugate of deoxycholic acid, a distinguishabletaurine conjugate of deoxycholic acid, a distinguishable lithocholicacid, a distinguishable glycine conjugate of lithocholic acid, adistinguishable taurine conjugate of lithocholic acid, and aphysiologically acceptable salt of a distinguishable cholate compound.21. The method of claim 20, wherein the distinguishable cholate compoundis a distinguishable cholic acid or a distinguishable chenodeoxycholicacid.
 22. The method of claim 20, wherein the distinguishable cholatecompound is a stable isotope labeled cholate compound.
 23. The method ofclaim 22, wherein the stable isotope labeled cholate compound is astable isotope labeled cholic acid or a stable isotope labeledchenodeoxycholic acid.
 24. The method of claim 1, wherein thedistinguishable bile acid exhibits a high hepatic extraction of greaterthan 70% in first pass through the liver of a healthy subject.
 25. Themethod of claim 1, wherein the distinguishable bile acid exhibitsremoval from the blood of the subject exclusively by the liver.